Ordered biological nanostructures formed from chaperonin polypeptides

ABSTRACT

The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of prior U.S. provisional applicationNo. 60/340,538, titled “Ordered Biological Nanostructures Formed FromExtremophillic Heat-Shock Proteins,” filed on Nov. 8, 2001, which ishereby incorporated by reference in its entirety, including drawings.

ORIGIN OF THE INVENTION

The invention described herein was made by an employee(s) of the UnitedStates Government and may be manufactured and used by or for theGovernment for governmental purposes without payment of the royaltiesthereon or therefor.

1. Field of the Invention

The following application relates to nanotemplates, nanostructures,nanoarrays and nanodevices formed from wild-type and mutated chaperoninpolypeptides, methods of producing such compositions, methods of usingsuch compositions and particular chaperonin polypeptides that can beutilized in producing such compositions.

2. Background of the Invention

The controlled organization of inorganic materials intomulti-dimensional addressable arrays is the foundation for both logicand memory devices, as well as other nonlinear optical and sensingdevices (Zhirnov et al., 2001, Computer 34, 34-43, Xia et al., 2000,Adv. Mater. 12, 693-713). Many of these devices are currently fabricatedusing lithographic patterning processes that have progressivelydeveloped toward greater integration densities and smaller sizes. Atsubmicron scales, however, conventional lithographic processes areapproaching their practical and theoretical limits. At scales below 100nm, ion and electron beam lithography becomes prohibitively expensiveand time consuming, and more importantly, at these scales quantumeffects fundamentally change the properties of devices (Sato et al.,1997, J. Appl. Phys. 82, 696).

Nanoscale templates for constrained synthesis, in situ deposition, ordirect patterning of nanometer scale inorganic arrays are beingdeveloped using both artificial and natural materials. Artificialmaterials such as microphase separated block copolymers (Park et al.,2001, Appl. Phys. Lett. 79, 257-259) and hexagonally close-packedspheres (Hulteen et al., 1995, J. Vac. Sci. Technol. A, 1553-1558) havebeen used for nanoscale fabrication. Natural materials such as DNA(Richter et al., 2000, Adv. Mater. 12, 507-510; Keren et al., 2002,Science 297, 72-75), bacterial and archaeal surface layer proteins(S-layer proteins) (Sleytr et al., 1999, Angew. Chem. Int. Ed. 38,1034-1054; Douglas et al., Appl. Phys. Lett. 48, 676-678; Hall et al.,2001, CHEMPHYSCHEM 3, 184-186), virus capsids (Shenton et al., 1999,Adv. Mater. 11, 253-256; Douglas et al., 1999, Adv. Mater., 679-681;Douglas et al., Nature 393, 152-155; Wang et al., 2002, Angew. Chem.Int. Ed. 41, 459462), phage (Lee et al., 2002, Science 296, 892-895),and some globular proteins (Yamashita, I., 2001, Thin Solid Films 393,12-18) have been used as templates and in other nanoscale applications.

Various nanometer scale objects, including arrays of nanoparticlesformed by non-conventional methods are being explored for use as viablealternatives to standard lithographically patterned devices. Individualnanoparticles, also known as quantum dots (QDs), have been shown tobehave as isolated device components such as single electron transistors(Likharev, K. K., 1999, Proc. IEEE 87, 606-632; Thelander et al., 2001,Appl. Phys. Lett. 79, 2106-2108). Theoreticians have postulated thattwo-dimensional arrays of QDs with nanoscale resolution could form thebasis of future generations of electronic and photonic devices. Thefunction of these devices will be based on phenomena such as coulombcharging, inter-dot quantum tunneling and other coherent propertiesderived from the electronic consequences of confinement and nanoparticlesurface area to volume ratios (Maier, S. A. et al., 2001, Adv. Mater.13, 1501-1505; Maier et al., Phys. Rev. B 65, 193408; Zrenner, A. etal., 2002, Nature 418, 612-614; Berven et al., 2001, Adv. Mater. 13,109-113).

Traditional techniques for patterning ordered arrays of materials ontoinorganic substrates and manufacturing devices currently used are ionbeam lithography and molecular beam epitaxy. These techniques possessinherent limitations due to the use of polymeric light masks for patternformation, however, there is a theoretical limitation of patterning thatcould ultimately limit the processes in the hundreds of nanometers.

While there are strong incentives to develop nanoscale architectures,these developments require alternate fabrication methods and newinsights into the behavior of materials on nanometer scales (Nalwa, H.S., 2000, Handbook of materials and nanotechnology, Academic Press, SanDiego).

3. SUMMARY OF THE INVENTION

The invention provides a method of forming higher order structurescomprising at least one mutated chaperonin polypeptide. Such higherorder structures include nanotemplates, nanostructures, nanoarrays andnanodevices.

The invention provides a nanotemplate comprising chaperoninpolypeptides, wherein at least one polypeptide is a mutated polypeptide.The invention also provides higher order structures comprising at leastone mutated chaperonin polypeptide and at least one nanoparticle orquantum dot, including nanostructures, nanoarrays and nanodevices. Ananoarray comprises an ordered array of the nanostructures. A nanodevicecomprises at least one nanotemplate, at least one nanostructure, atleast one nanoarray or some combination thereof.

The invention also provides a method of forming the nanostructures,nanoarrays and nanodevices. The steps include (a) adding one or morenanounits to a surface, where such nanounits include eithernanotemplates or a mixture of nanotemplates and wild-type chaperonins,and (b) adding or synthesizing in situ one or more nanounits comprising(i) at least one nanoparticle, (ii) at least one quantum dot, or (iii) acombination of (i) and (ii) to said surface and (c) ) if necessary,removing any unbound nanounits. The steps are repeated any number oftimes in any sequence to form the nanostructures, nanoarrays andnanodevices.

The invention provides variants of chaperonin polypeptide subunitsthrough selective mutation of the chaperonin polypeptide sequence. Themutant chaperonin comprises one more mutated chaperonin polypeptidesequences. The invention provides chaperonin polypeptide variants withone or more point mutations. The invention provides chaperoninpolypeptide variants with one or more residues or sequence of residuesinserted or deleted. The polypeptide sequences inserted are designed tobind nanoscale materials such as nanoparticles and quantum dots, or tobind only to specific surfaces. The invention also provides formutations to the N- and C-termini, including deletion of the terminus orinsertion of a sequence. In a specific embodiment, the chaperoninpolypeptides are HSP60 heat shock proteins.

The invention also provides a method for forming a mutated chaperonin.The steps include modifying at least one protein residue of a chaperoninpolypeptide by positioning a mutation to form one or more mutatedchaperonin polypeptides, and assembling the one or more mutatedchaperonin polypeptides to form a mutated chaperonin.

By genetically engineering a polypeptide that self-assembles intoregular double-ring structures known as chaperonins, the presentinvention teaches methods of directing the organization ofnanoparticles, e.g., preformed metal and semiconductor nanoparticles,and quantum dots (QDs), into nanostructures, nanoarrays and nanodevices.The present invention teaches methods of assembling mutated chaperoninpolypeptides into structures that function, for example, asnano-vessels, nano-wires, nanotemplates, nano-fabrics, and nanoarrays,e.g., DNA, RNA and/or peptide or polypeptide nanoarrays.

The present invention further provides methods for manufacturingnanodevices, e.g., microelectronics, using chaperonins, in particular,mutant chaperonins comprising at least one mutated chaperoninpolypeptide. In one embodiment, the mutant chaperonins comprise at leastone mutant extremophillic HSP60 (heat-shock protein).

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an end and side view of a HSP60 chaperonin at 2.3 Åresolution. The outlined region of the side view shows a single subunitof HSP60.

FIGS. 2A-2R show the protein sequence alignment of S. shibatae TF55 betasubunit (SEQ ID NO: 1), bacterial E. coli GroEL (SEQ ID NO:2),thermosome T. acidophilum beta subunit (SEQ ID NO:3), cyanobacterialsynechococcus HSP60 (SEQ ID NO:4), M. acetivorans HSP60-4 (SEQ ID NO:5),M. tuberculosis HSP65 (SEQ ID NO:6), thermosome A. pernix alpha subunit(SEQ ID NO:7), thermosome M. mazei alpha subunit (SEQ ID NO:8),mitochondrial A. thaliana HSP60 (SEQ ID NO:9), yeast TCP1 alpha subunit(SEQ ID NO: 10), human mitochondrial HSP60 (SEQ ID NO: 11), mousemitochondrial HSP60 (SEQ ID NO:12), human TCP1 alpha subunit (SEQ IDNO:13), mouse TCP1 alpha subunit (SEQ ID NO:14), and the consensus (SEQID NO:15). Identical residues are enclosed in a dot-dashed box, blocksof similar residues are enclosed in a solid box, and conservativematches are enclosed in a dashed box.

FIG. 3 shows a structural alignment of the archaeal chaperonin(thermosome) and the bacterial chaperonin (GroEL), indicating thestructural similarities between group I and group II chaperonins. Theblack areas of the structural alignment indicate where the features ofthe two chaperonin subunits overlap.

FIG. 4 shows the detailed structure of a Group II chaperonin subunit.

FIGS. 5A-D shows individual HSP60 (heat-shock protein) chaperonins andfilaments as observed in the electron microscope.

FIGS. 6A and 6B show the organization of HSP60 rings into 2-dimensionalcrystals on a metal grid coated with lipid (6A) and filament bundlesarranged on a bed of rings (visible as spots in background) (6B).

FIGS. 7A-E show the assembly of engineered HSP60s (heat-shock proteins)into nanotemplates for the production of nanoarrays comprising nanoscalematerials such as nanoparticles. templates.

FIGS. 8A-D show gold nanoparticles binding to engineered chaperonins andchaperonin nanotemplates.

FIGS. 9A-D show semiconductor QD nanoarrays.

FIGS. 10A-D show the formation of a nanoarray of gold nanoparticles.FIG. 10(D) shows XEDS spectra of bare carbon film (solid line) and thegold nanoparticle nanoarray (dashed line) from the probed area outlinedby a circle in FIG. 10(B), as indicated by the arrow.

FIGS. 11A-C show HAADF STEM imaging of a nanogold array.

FIG. 12 shows a control experiment showing DIC (left) and fluorescent(right) images of non-cys-mutated chaperonin crystals after incubationwith CdSe—ZnS QDs.

FIG. 13 shows an Energy Filtered TEM thickness map of a typical 2Dprotein crystal.

FIG. 14 illustrates steps in the formation of an ordered nanoarray ofnanoparticles on a substrate.

FIG. 15 shows the protein sequence alignment of S. shibatae TF55 alphasubunit (SEQ ID NO: 39), beta subunit (SEQ ID NO: 1) and gamma subunit(SEQ ID NO: 38).

FIGS. 16A and 16B show the DNA sequence (SEQ ID NO: 37) and amino-acidsequence for S. shibatae gamma subunit (SEQ ID NO: 38).

5. DETAILED DESCRIPTION OF THE INVENTION

All patents cited in the specification are hereby incorporated byreference in their entirety. In the case of inconsistencies, the presentdisclosure, including definitions and terminology, will prevail.

5.1 Terminology

The term “nanotemplate” as used herein, unless otherwise indicated,refers to a composition comprising one or more chaperoning, wherein atleast one chaperonin is a mutant chaperonin comprising at least onemutated chaperonin polypeptide. In one embodiment, a nanotemplate is acomposite of both wild-type and mutant chaperoning. It is noted that theterms “chaperonin polypeptide” chaperonin subunit” and “chaperoninpolypeptide subunit,” are utilized herein interchangeably.

The term “nanostructure” as used herein, unless otherwise indicated,refers to a composition comprising one or more nanotemplates and one ormore nanoscale materials, such as nanoparticles and/or quantum dots.

The term “nanoarray” as used herein, unless otherwise indicated, refersan ordered arrangement of nanotemplates and/or nanostructures.

The term “nanotemplate” as used herein, unless otherwise indicated,refers to a device comprising at least one nanotemplate, at least onenanostructure and/or at least one nanoarray. Exemplary devices include,but are not limited to, electronic, semiconductor, mechanical,nanoelectromechanical, magnetic, photonic, optical, optoelectronic orbiomedical devices.

The term “nanounit” as used herein, unless otherwise indicated, refersany of the components or “basic building blocks” of a nanostructure,including, for example, a nanscale object, such as a nanoparticle or aquantum dot, a nanotemplate, and a wild-type chaperonin or chaperoninpolypeptide, or a mutant chaperonin or chaperonin polypeptide.

5.2 Detailed Description of the Preferred Embodiments

The following application relates to nanotemplates, nanostructures,nanoarrays and nanodevices formed from wild-type and mutated chaperoninpolypeptides, methods of producing such compositions, methods of usingsuch compositions and particular chaperonin polypeptides that can beutilized in producing such compositions.

Chaperonins

The compositions and devices of the invention, e.g., the nanotemplates,nanostructures, nanoarrays and nanodevices of the invention, comprise,unless otherwise indicated, at least one mutant chaperonin, whichcomprises at least one mutant chaperonin polypeptide. In manyembodiments, the compositions can further comprise chaperonins that donot contain mutant chaperoning. Moreover, in many embodiments, themutant chaperonins can further comprise non-mutant, that is, wild-typechaperoning. Non-limiting examples of chaperonins and chaperoninpolypeptides that can be utilized as part of the methods andcompositions of the present invention are described herein. Non-limitingexamples of mutant chaperonins and mutant chaperonin polypeptides thatcan be utilized as part of the methods and compositions of the presentinvention are described hereinbelow, in the following section.

Chaperonins (also referred to herein as “cpn60s”) are double-ringedstructures comprising approximately 60 kDa (±5 kDa) proteins (see, e.g.,Hartl et al., 2002, Science 295, 1852-8). In nature, chaperonins areubiquitous and essential subcellular structures comprising 14, 16, or 18protein subunits, arranged as two stacked rings approximately 16 to 18nm tall by approximately 15 to 17 nm wide, depending on their species oforigin. FIG. 1 illustrates an end and side view of a chaperonin thatcomprises 16 subunits, i.e., eight subunits per ring.

The sequence and three dimensional structural similarities betweenchaperonins and chaperonin polypeptides allows any chaperoninpolypeptides and chaperonins to routinely be utilized as part of thecompositions and devices of the present invention. In addition, suchsimilarities allow any chaperonin polypeptides to routinely be able toused to derive the mutant chaperonin polypeptides that comprise thecompositions and devices of the invention. The sequence and threedimensional structural similarity of the subunits among the differenttypes of chaperonins, which is illustrated by the sequence alignmentdepicted in FIGS. 2A-2R and the structural overlap as illustrated in arepresentative comparision depicted in FIG. 3, provides the basis forthe formation of the nanotemplates, nanostructures, nanoarrays andnanodevices of the invention.

Chaperonins have been classified into two groups, Group I and Group II,based on sequence and structural comparisons. (See, e.g., Trent et al.,1991, Nature 354, 490493; Horwich et al., 1993, Phil. Trans R. Soc.Lond. 339, 313-326). Group I or Group II chaperonins or chaperoninpolypeptides, or mutant chaperonins comprising at least one mutant GroupI or Group II chaperonin polypeptide, can be utilized as part of thecompositions and devices of the present invention. In one embodiment,the chaperonins or mutant chaperonins comprise Group I chaperoninpolypeptides and/or mutant chaperonin polypeptides. In anotherembodiment, the chaperonins and/or mutant chaperonins comprise Group IIchaperonin polypeptides and/or mutant chaperonin polypeptides. In yetanother embodiment, the chaperonins or mutant chaperonins comprise GroupI and Group II chaperonin polypeptides and/or mutant chaperoninpolypeptides.

Group I chaperonins are from bacteria and the bacterial-derivedorganelles of Eukarya (mitochondria and chloroplasts), while group IIchaperonins are from Archaea and eukaryotic cytosol. See, e.g., U.S.Pat. No. 5,428,131 to Trent et al. that describes the expression ofendogenous, wilde-type TF155 S. shibatae, and provides a comparison of agroup I chaperonin (GroEL) to the group II chaperonin TF55.

Wild-type Group I chaperonins are composed of seven subunits in each ofthe two rings of the double-ring structure. The wild-type cpn6oproteins, which comprise about 550 to about 580 amino acid residues,have been described by different names in different species, including,but not limited to Escherichia coli GroEL protein, Cyanobacterial groELanalogues, Mycobacterium tuberculosis and leprae 65 Kd antigen, Coxiellaburnetti heat shock protein B (gene htpB), Rickettsia tsutsugamushimajor antigen 58, Chlamydial 57 Kd hypersensitivity antigen (gene hypB),Chloroplast RuBisCO subunit binding-protein alpha and beta chains,Mammalian mitochondrial matrix protein P1 (mitonin or P60), and YeastHSP60 protein. Any of these chaperonins, or mutants thereof, can, forexample, be utilized as part of the compositions and devices of thepresent invention.

In one embodiment, e.g., when utilizing Group I chaperoning, chaperoninpolypeptides, and/or mutant chaperonins and/or mutant chaperoninpolypeptides, a cochaperonin can be utilized in forming the higher orderstructures of the invention. As such, in one example of such anembodiment, a composition or device of the invention further comprises acochaperonin. Cochaperonins are well known to those of skill in the art.See, e.g., Harris et al., 1995, J. Structural Biol. 115, 68-77). Inanother, non-limiting example of such an embodiment, a cochaperonin canbe utilized in producing nanofilaments. For example, the cpn60 in thebacterium E. coli (GroEL) in nature is associated with a single ringstructure composed of 10 kDa proteins (co-chaperonin or cpn10) called“GroES.” As such, a GroES polypeptide represents an exemplary,non-limiting species of cochaperonin that can be utilized in conjunctionwith Group I chaperoning, e.g., GroEL or GroEL-derived chaperoning,chaperonin polypeptides, and/or mutant chaperonins or chaperoninpolypeptides. In different embodiments of the invention, thecompositions, e.g., nanotemplates or nanostructures, are formed from oneor more chaperonins with the cochaperonin on one or both ends of thechaperonin.

Group II chaperonins are composed of identical or diverse subunitsarranged in rings of eight or nine subunits, depending on the organism.In the yeast Saccharomyces cerevisiae, for example, there is evidencefor eight different subunits in each ring (Lin et al., 1997, Proc. Natl.Acad. Sci. USA 94, 10780-10785). Among the Archaea some thermophilicmethanogens (e.g., Methanopyrus kandleri, Methanococcus jannaschii,Methanococcus thermolithotrophicus) have chaperonins with identicalsubunits (Furutani et al., 1998, J. Biol. Chem. 273, 28399-28407), whilein the mesophilic methanogen Methanosarcina acetivorans there are fivedifferent subunits (Galagan et al., 2002, Genome Research 12, 532-542).Of the 50 archaeal chaperonin sequences in the databases most have >40%amino acid sequence identity. Any of these chaperoning, or mutantsthereof, can, for example, be utilized as part of the compositions anddevices of the present invention.

The majority of group II chaperonins in Archaea have eight subunits perring and are referred to as “thermosomes” (Klumpp, M., and Baumeister,W., 1998, FEBS Letters 430, 73-77), but the chaperonins in thethermoacidophilic Archaea in the familiy Sulfolobales have nine subunitsper ring (Trent et al., 1991, Nature 354, 490-493; Marco et al., 1994,FEBS 341, 152-155). These Sulfolobus octadecameric chaperonins arereferred to as “rosettasomes” (Kagawa et al., 1995, J. Mol. Biol. 253,712-725) to distinguish them from thermosomes. Other examples ofthermosomes include chaperonins from Pyrodictium occultuim, Thermoplasmaacidophilum and Methlanopyrus kandleri (Ellis et al., 1998, J. Struc.Biol. 123, 30-36). It has previously been reported that rosettasomes arecomposed of two types of HSP60s known as TF55 α and β that TF55 α and βare among the most abundant proteins in S. shibatae grown at optimaltemperatures (75-83° C.), and that their synthesis increases atheat-shock temperatures (85-88° C.) (Kagawa et al., 1995, J. Mol. Biol.253, 712-725). A third related subunit of S. shibatae, has also beenidentified by sequence analyses (Archibald et al., 1999, Current Biology9, 1053-1056). Sequence information from S. solfataricus (Charlebois etal., 1998, Current Opinion in Microbiology 1, 584-588) allowed TF55alpha, beta, and gamma expression to be predicted based on codon usage(Karlin et al., 2001, J. Bacteriol. 183, 5025-5040). Chaperonins fromeukaryotic cytosol are referred to as “TCP1,” which identifies one ofthe proteins comprising the ring structure, “TriC” which means TCP1 ringchaperonin, or “CCT” which means chaperonin containing TCP1. Any ofthese chaperoning, or mutants thereof, can, for example, be utilized aspart of the compositions and devices of the present invention.

In one embodiment, the chaperonins comprise HSP60s (heat shockproteins), which are proteins induced by heat stress.

FIGS. 2A-2R show protein sequence alignments covering a representativeset of Groups I (bacteria) and Group II (archaea and eukarya)chaperoning. The protein sequence are sequences for S. shibatae TF55beta subunit (SEQ ID NO: 1), bacterial E. coli GroEL (SEQ ID NO:2),thermosome T. acidophilum beta subunit (SEQ ID NO:3), cyanobacterialsynechococcus HSP60 (SEQ ID NO:4), M. acetivorans HSP60-4 (SEQ ID NO:5),M. tuberculosis HSP65 (SEQ ID NO:6), thermosome A. pernix alpha subunit(SEQ ID NO:7), thermosome M. mazei alpha subunit (SEQ ID NO:8),mitochondrial A. thaliana HSP60 (SEQ ID NO:9), yeast TCP1 alpha subunit(SEQ ID NO:10), human mitochondrial HSP60 (SEQ ID NO:11), mousemitochondrial HSP60 (SEQ ID NO:12), human TCP1 alpha subunit (SEQ IDNO:13), mouse TCP1 alpha subunit (SEQ ID NO:14), and the consensus (SEQID NO:15). White letters on a black background, solid lines, and dashedlines surround the regions of the sequence alignment containingidentical residues, a block of similar residues, and conservativematches, respectively.

For purposes of wild-type chaperonins and chaperonin polypeptides, suchsequence similarity serves to illustrate that fact that any chaperoninor chaperonin polypeptide routinely can be utlized as part of thecompositions and devices of the present invention, either alone orcombination. For purposes of mutant chaperonins and chaperoninpolypeptides, as discussed in detail in the next section, such sequencesimilarity serves to provide teaching that allows for routinemanipulation of sequences in producing and modifying mutant chaperoninpolypeptides that can become part of mutant chaperonins in thecompositions and devices of the present invention.

While group I chaperonins can have greater than 50% sequence identity,sequence identity among Group II chaperonins can be on the order of lessthan 33%. Despite the sequence variations among the cpn60 subunits fromthe different species, however, group I and group II cpn60 subunitsshare significant structural similarity. FIG. 3 shows a structuralcomparison between a subunit of the archaeal (Themroplasma acidophilum)thermosome and the bacterial (E. coli) GroEL chaperonins. The alignmentwas performed using an algorithm based on the iterative dynamicprogramming approach as outlined Gerstein, M. & Levitt, M., ProteinScience 7, 445-456, 1998; and Gerrstein, M. & Levitt, M, Proc. ofISMB-96, pp. 59-67, 1996.

For purposes of wild-type chaperonins and chaperonin polypeptides, suchthree dimensional structural similarity serves to illustrate that factthat any chaperonin or chaperonin polypeptide routinely can be utilizedas part of the compositions and devices of the present invention, eitheralone or combination. For purposes of mutant chaperonins and chaperoninpolypeptides, as discussed in detail in the next section, such sequencesimilarity serves to provide teaching that allows for routinemanipulation of sequences in producing and modifying mutant chaperoninpolypeptides that can become part of mutant chaperonins in thecompositions and devices of the present invention.

The two subunits exhibit very similar structures, in that both possessan equitorial, an intermediate and an apical region. Even though thesetwo examples of cpn60 subunits are farther apart by sequence than mostcpn60 subunits, as evidenced by the very little similarity in theirsequence alignments (see FIGS. 2A-2R), the crystal structures for eachreveal that they share considerable structural identity—most allhelical, sheet, and random coil regions correspond, as shown in black inthe center panel. Variations in structure are tolerated in the apicaldomain, as evidenced by the loop of the thermosome, while the equitorialdomains adopt similar conserved folding motifs.

It is noted that, while the chaperonins observed to date comprise seven,eight or nine subunits per ring, the present invention provides methodsand compositions of exploiting chaperonins with any number of subunitsper ring for the compositions, e.g., nanotemplates, nanostructures, andnanoarrays, and nanodevices of the invention.

Chaperonins from the different species can comprise only a single typeof subunit or they can have different types of subunits (e.g., archealchaperonins comprising alpha, beta, gamma, etc.). These subunits arecalled alpha subunits, beta subunits, or gamma subunits, due to somedifferences in the protein sequences of the subunits of a given species.As is known to one of ordinary skill in the art, in some species yetmore varieties of subuits exist. The structure of chaperonins Ellis etal., 1998, J. Struc. Biol. 123, 30-36 describes a chaperonin fromSulfolubus solfataricus with a 2:1 ratio of alpha:beta subunitcomposition of the nine-membered ring (rosettasomes). The presentinvention provides means of assembling chaperonins from only a singletype of wild-type or mutated chaperonin polypeptide, or from variousproportions of the different wild-type or mutated chaperoninpolypeptides.

In a specific embodiment, HSP60s (heat-shock proteins) in organismsliving at high temperatures, called “thermophiles,” are the source ofthe wild-type and mutated chaperonin polypeptides of the presentinvention. These proteins are present in all organisms and are among themost abundant proteins in extreme thermophiles, e.g., in one of thehighest temperature thermophiles Pyrodictium occultum, they reportedlyaccount for 73% of total protein (Phipps et al., 1991, The EMBO Journal10(7), 1711-1722).

Chaperonin and mutant chaperonin polypeptides can routinely be expressedusing standard techniques well known to those of skill in the art. Forexample, sequences encoding the chaperonin polypeptide and/or mutantchaperonin polypeptide can be introduced into a host cell, e.g., aprokaryotic, for example, an E. coli or Salmonella host cell,eukaryotic, for example, a yeast or mammalian host cell, and expressedand isolated using standard recombinant techniques.

In a non-limiting example, a secquence encoding a thermostablechaperonin, e.g. a thermostable HSP60, can be transferred into E. coliand grown at temperatures standard for the cell. The expressedpolypeptide can then be easily purified from E. coli proteins by heatingand centrifugation. The thermolabile E. coli proteins precipitateleaving the thermostable polypeptide greater than 90% pure after acentrifugation. Another advantage is that HSP60 nanotemplate structuressuch as rings, tubes, and filaments (to be described in detail below)bind to DNA and RNA using the method of “gel shift,” to proteins by themethod of autoradiography, and to liposomes and lipid monolayers.

Mutant Chaperonins

The present invention provides methods for forming variants ofchaperonin polypeptides through selective mutation of the polypeptide,and then exploiting the ability of these variants to self-assemble intohigher-order structures under various conditions for forming thecompositions and devices, e.g., nanotemplates, nanostructures,nanoarrays and nanodevices, of the invention.

The compositions and devices of the invention, e.g., the nanotemplates,nanostructures, nanoarrays and nanodevices of the invention, comprise,unless otherwise indicated, at least one mutant chaperonin, whichcomprises at least one mutant chaperonin polypeptide. Non-limitingexamples of mutant chaperonins and mutant chaperonin polypeptides thatcan be utilized as part of the methods and compositions of the presentinvention are described herein.

In referring to mutant chaperonins and mutant chaperonin polypeptides,the term “mutant” refers to a difference relative to what is considereda wild-type sequence. Representative, non-limiting examples of wild-typechapernin polypeptide sequences are presented in FIGS. 2A-2R. Inaddition, in one embodiment, a mutant chaperonin polypeptide is onethat, when present in a cell or organism, yields an observable phenotypethat differs from the phenotype observed in its absence, that is, whenonly a corresponding wild-type sequence is present. Generally, a mutantchaperonin sequence refers to a sequence that does not occur in natureat a greater than 10% (+/−10%) allelic frequency, as measured bystandard methods and available data. For example, an example of a mutantS. shibatae chaperonin polypeptide is one that is expressed by an allelethat is present in the organism at no greater than 10% (+/−10%) alleicfrequency.

As discussed above, the sequence and three dimentsional structuralsimilarities between chaperonins and chaperonin polypeptides allows anychaperonin polypeptides and chaperonins to routinely be utilized as partof the compositions and devices of the present invention. Moreover, suchsimilarities allow any chaperonin polypeptides to routinely be able toused to derive the mutant chaperonin polypeptides that comprise thecompositions and devices of the invention. The sequence and threedimensional structural similarity of the subunits among the differenttypes of chaperoning, which is illustrated by the sequence alignmentdepicted in FIGS. 2A-2R and the structural overlap as illustrated in arepresentative comparision depicted in FIG. 3, provides the basis forthe formation of the nanotemplates, nanostructures, nanoarrays andnanodevices of the invention.

Further, the details of the structure of chaperonins can be solved atatomic-resolution (2.3-2.8 Å) (See, e.g., FIG.; 1 and Xu, Z. et al.,1997, Nature 388, 741-750; and Ditzel, L., J. Lowe, et al., 1998, Cell93, 125-138). This provides detailed information about the location ofevery atom of every amino acid in the double ring structure (e.g., FIG.4), and can be used to routinely choose chaperonin sites formodification and can routinely assess the properties of chaperoning, inparticular, mutant chaperoning.

Utilizing the sequence and three dimensional structural similaritiesamong chaperonins and chaperonin polypeptides, as well as the ability tosolve at atomic-resolution the structure of particular chaperonins andchaperonin polypeptides, the structure of the chaperonin polypeptidescan be manipulated to influence, for example, their assembly, strength,and binding properties, as well as the assembly, strength and bindingproperties of the resulting chaperonins and, in turn, compositions anddevices comprising the chaperoning.

Such structural similarities can be utilized in a number of differentways in choosing appropriate mutants. For example, a mutant in onespecies that exhibits a desirable characteristic can be introduced intoa corresponding position in another chaperonin by utilizing the sequencesimilarity and/or the three dimensional structural similarity betweenthe chaperoning. In one such embodiment, for example, the mutant S.shibate sequences successfully utilzed in the examples presented belowcan routinely be introduced into other chaperonin polypeptides by thesetechniques, and the resulting mutant chaperonin polypeptides can be usedin the compositions and devices of the invention.

Standard methods well known in the art which allow changing specificamino acids in chaperonin polypeptides, such as the method ofsite-directed mutagenesis, regions of the subunits can be modified, andthe resulting chaperonin polypeptides can routinely be tested for theirability to produce chaperonins and, for example, nanotemplates,nanostructures, nanoarrays and nanodevices, e.g., their ability toassemble into tubes and filaments can be tested. In one embodiment, forexample, amino acid tails can be attached to chaperonin polypeptidesubunits that do not inhibit their ability to assemble into rings andtubes, and that allow the binding of various nanoscale materials, suchas metals, at various locations of the chaperonins, including inside thechaperonin structure. In one embodiment, one of the three HSP60 subunits(beta) from Sulfolobus shibatae, an organism that lives in geothermalhot-springs and grows at temperatures of up to 85° C./pH 2.0 is used toform mutant chaperonins. The chaperonins in S. shibatae areoctadecameric with nine subunits per ring. FIG. 15 shows the proteinsequence alignment of S. shibatae TF55 alpha subunit (SEQ ID NO: 39),beta subunit (SEQ ID NO: 1) and gamma subunit (SEQ ID NO: 38). The betasubunit can be chosen for a particular application based on such factorsas its thermostability, which makes it easy to purify as a recombinantprotein, and the availability of sequence and structural information,which can guide the genetic manipulations.

In general, the chaperonin subunits have many regions that canaccommodate additions or deletions in each of their threedomains-equatorial, intermediate, and apical domains, as illustrated inFIG. 3. FIG. 4 shows the detailed structure of a Group II chaperoninsubunit that can be used in making a choice of mutations. The mutationscan be performed to engineer one or more specific binding sites atdifferent locations on a chaperonin for the attachment of a quantum dotor a nanoparticle, as is described in greater detail below, or for theattachment of different types of molecules or polypeptides. Themutations can modify the dimensions of the resulting chaperonin, such aslength, inner pore diameter, outer diameter, etc., or it can beperformed to present specific binding sites on the apical, intermediateor equitorial regions of the chaperonin. The choice of mutation dependson the desired structure for the different applications of the presentinvention, including the formation of nanotemplates, nanostructures,nanoarrays and nanodevices.

The choice of mutations to make depends on the desired structure of theresulting chaperonin, and can routinely be ascertained. In a specificembodiment, the mutated chaperonin polypeptide subunits include onesthat assemble into higher order structures with less than seven subunitsper ring or more than nine subunits per ring. Mutations can be made tothe subunit sequence such that the resulting subunit variants assembleinto a structure with any number of subunits per ring. Mutationsintroduced that that change in number of subunits per ring can, forexample, be used to modify the diameter of a resulting ringnanostructure.

Factors that affect the choice of which chaperonin polypeptides tomanipulate (e.g., from what species, which subunit(s), etc.), and whatmutations are to be made to them, include the desired dimensions, i.e.,length, pore diameter, and outer diameter, of the resulting chaperoninproduct, or introduction of a selective binding site anywhere on thepolypeptide. The subunits of both group I and group II chaperonins willtolerate a point mutation at any position. When sequence alignments areused in determining mutation positions, mutations at similar,non-identical residues, as determined by sequence alignment, beingpreferred, and non-conserved positions, as determined by sequencealignment being more preferred. When three dimensional structuralalignments are used in determining mutation positions, a structuralalignment of chaperonin subunits, such as that of FIG. 3, can serve as aguide in deciding where on the subunit to perform the mutation. Theloops and turns from the two structures that do not directly superimposecan be choices of points to perform mutations, including deletions andinsertions. In addition, the N- and/or C-termini of the polypeptides aregenerally amenable to manipulation.

In one embodiment, a choice of deletion of the amino acid loop at theapical domain of a group II chaperonin is made through comparison of thestructural alignment of FIG. 3, and with the observation that theloopless group I chaperonin subunit assembles into thedouble-ring-structure of the chaperonin. In another embodiment, the N-or C-terminus is removed. In yet another embodiment, the N- orC-terminus is modified by inserting a sequence. The sequence can beinserted for binding specificity, such as by introducing cysteine ortyrosine which can be modified chemically.

In a specific embodiment, the mutant chaperonin comprises one moremutated chaperonin polypeptide sequences with one or more pointmutations. An exemplary point mutation in TF55-beta from Sulfolobusshibatae results from residue 298 being changed from cysteine to alanineand residue 270 changed from glutamine to cysteine. In anotherembodiment, the mutant chaperonin comprises one more mutated chaperoninpolypeptide sequences with one or more sequences deleted. An exemplarydeletion in TF55-beta results from Solfolobus shibatae with residues 254to 281 deleted. In another embodiment, the mutant chaperonin comprisesone or more mutated chaperonin polypeptide sequences with one or morepolypeptide sequences inserted. An exemplary insertion in TF55-betaresults from Solfolobus shibatae with peptides that possess bindingspecificity inserted. As discussed above; corresponding mutations can beroutinely introduced into any other chaperonin polypeptide.

In another embodiment, the peptides are designed to bind nanoscalematerials such as nanoparticles and quantum dots. In yet anotherembodiment, the peptides are designed to bind only to specific surfaces.Still other modifications can also be made in the equatorial domainsthat include deletions, substitutions and additions to the N- andC-termini with little effect on the formation of chaperonins ornanotemplates such as filaments. For example, up to about 5, 10, 15, 20,25, or 30 amino acids of the N- and/or C-terminus of the chaperoninpolypeptide can be modified, e.g., deleted. For example, GroEL can bemodified by removing up to about 27 amino acids from the C-terminuswithout impairing its ability to assemble into double rings.

Additional references that describe possible mutations of specificresidues of the polypeptides are contained in the review article Fentonet al., 1997, Protein Science 6, 743-760.

The sequence alignment of FIGS. 2A-2R indicates that the regions thathave been manipulated in S. shibatae also exist in other species.Whatever mutations have been successfully made in one species may besuccessfully others species, whether bacterial, other archea or eukarya.The corresponding regions of the sequence alignments can therefore serveas a guide in choice of manipulations to produce variants in otherspecies, combined with the knowledge of the region of the chaperoninsubunit that the given mutated sequence is located. A successfulmutation of the chaperonin polypeptide from any given species isindicated if the mutated chaperonin polypeptide retains its ability toassemble into the higher order structures of the invention, includingthe nanotemplates, nanostructures, nanoarrays and nanodevices.

In a specific embodiment, guided by structural information, the betasubunit of Sulfolobus shibatae is genetically modified to add chemicallyreactive sites without destroying its ability to assemble intochaperonins and 2D crystals. While a detailed three-dimensionalstructure of S. shibatae beta is not known, X-ray structures forhomologous chaperonin subunits are known (See, e.g., Xu et al. AndDiztel et al., supra.). Detailed transmission electron microscopic (TEM)analyses of S. shibatae chaperonins have also been reported (Trent etal., 1997, Proc. Nat. Acad. Sci 94, 5383-5388). Using X-ray structuresof homologous subunits and TEM analyses of Sulfolobus chaperonins, ahypothetical three-dimensional model for the beta chaperon can beproduced, and used to guide genetic manipulations (See, e.g., Peitsch,M. C., 1995, Bio/Technology 13, 658; Guex, N., Peitsch, M. C., 1997,Electrophoresis 18, 2714; Guex, N., Diemand, A., Peitsch, M. C., 1999,TiBS 24, 364). At least two classes of beta mutants can be created usingsite-directed mutagenesis, many of which retain their ability toassemble into chaperonins that form 2D crystals (FIGS. 7B and 7D).

In two classes of beta mutants of S. shibatae, the single nativecysteine residue in beta can be changed to a nonreactive residue, forexample, an alanine residue, e.g., to prevent potential issues withfolding and with assembly of mutant subunits. A cysteine can then placedat different solvent-exposed sites. The thiols of these cysteines canprovide binding sites for soft metals including gold and zinc, asdescribed in greater detail below. In one class of beta mutants of S.shibatae, the exposed cysteine is placed near the tip of a 28 amino acidloop on the apical domain of beta, which in the assembled chaperoninprotrudes into the central cavity. This mutant chaperonin has a ring ofreactive thiols with a diameter of approximately 3 nm on both ends (FIG.7A). In the other class of beta mutants of S. shibatae, the protruding28 amino acid loop was removed and placed the exposed cysteine on theapical domain itself. The mutant chaperonin assembled from this subunithas a ring of reactive thiols with a diameter of approximately 9 nm andan open pore into its central cavity (FIGS. 7D, 7E).

The beta subunit of S. shibatae proves to have sufficient structuralplasticity in its apical domain to accommodate both the amino acidsubstitutions and deletions can be made without loss of its ability toform chaperonins and 2D crystals. Under reducing conditions both classesof beta mutants formed chaperonins that assembled into disk-shaped,hexagonally packed 2D crystals up to 20 μm in diameter (FIG. 7B and 7D),the crystalline lattice ordering of which is confirmed by fast Fouriertransformation (FFT) of the TEM images (FIG. 7D, inset).

With knowledge of the sequences of the group I or group II chaperoninpolypeptide, any number of mutations can be judiciously placed at one ormore areas of the apical, intermediate and/or equitorial domains of thechaperonin polypeptide. As evidenced by the sequence alignment of FIGS.2A-2B, the regions that have been manipulated in S. shibatae also existin other species. Whatever mutations work in one species can be made towork in others. These corresponding regions of the sequence alignmentscan therefore serve as a guide in choice of manipulations to producevariants in other species. Thus, the many different varieties of bindingsites that can be placed at different locations on a chaperonin can beexploited in the formation of the nanotemplates, nanostructures,nanoarrays and nanodevices of the present invention.

Formation of Chaperonins

The chaperonin polypeptide subunits are used to form the compositionsand devices, e.g., nanotemplates, nanostructures, nanoarrays andnanodevices, of the present invention. Sources of chaperonin genesinclude but are not limited to bacterial chaperonin genes encoding suchproteins as Gro ES/Gro EL; archaeal chaperonin genes encoding suchproteins as TF55, TF56, alpha, beta, gamma, and cpn60s; mammalianchaperonins such as Hsp60, Hsp10, TCP-1, cpn60 and the homologues ofthese chaperonin genes in other species (J. G. Wall and A. Pluckthun,Current Biology, 6:507-516 (1995); Hartl, Nature, 381:571-580 (1996)).Additionally, heterologous genomic or cDNA libraries can be used aslibraries to select or screen for chaperonins.

The sequences encoding the chaperonin polypeptides of interest(wild-type or mutated polypeptides) are incorporated into DNA expressionvectors that are well known in the art. These circular plasmidstypically contain selectable marker genes (usually conferring antibioticresistance to transformed bacteria), sequences that allow replication ofthe plasmid to high copy number in E. coli, and a multiple cloning siteimmediately downstream of an inducible promoter and ribosome bindingsite. Examples of commercially available vectors include the pET system(Novagen, Inc., Madison, Wis.) and Superlinker vectors pSE280 and pSE380(Invitrogen, San Diego, Calif.).

The steps in the self assembly of the chaperonins of the presentinvention can be achieved by methods that are well known in the art ofrecombinant DNA technology and protein expression in bacteria. First,the gene of interest is constructed and cloned into the multiple cloningsite. In some cases, additional genes are also cloned into the sameplasmid, for example, when the other polypeptide sequences are to beinserted. For example, restriction enzyme cleavage at multiple sites,followed by ligation of fragments, is used to construct deletions in thepolypeptide sequences as listed above. Alternatively, a single ormultiple restriction enzyme cleavage, followed by exonuclease digestion(EXO-SIZE, New England Biolabs, Beverly, Mass.), is used to delete DNAsequences in one or both directions from the initial cleavage site; whencombined with a subsequent ligation step, this procedure produces anested set of deletions of increasing sizes. Similarly, standard methodsare used to recombine DNA segments from different chaperonin polypeptidegenes, to produce genes for mutated chaperonin polypeptides. In general,these methods are also used to modify the N- or C-termini. Thus novelmutant polypeptides and combinations of polypeptides can be created toenable the formation of novel chaperonin polypeptide-based structures.

E. coli can serve as an efficient and convenient factory for thesynthesis of the protein subunits from a variety of sources, includingE. coli itself. In the next step, E. coli cells are transformed with therecombinant plasmid and the expression of the cloned gene is induced.The preferred hosts for production of the polypeptide is E. coli strainBL21(DE3) and BL21(DE3/pLysS) (available commercially from Novagen,Madison, Wis.), although other compatible recA strains, such asHMS174(DE3) and HMS174(DE3/pLysS) can be used. Transformation with therecombinant plasmid (Step 2) is accomplished by standard methods(Sambrook, J., Molecular Cloning, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y.; this is also the source for standard recombinantDNA methods used in this invention.) Transformed bacteria are selectedby virtue of their resistance to antibiotics e.g., ampicillin orkanamycin. The method by which expression of the cloned chaperoninpolypeptide is induced (Step 3) depends upon the particular promoterused. A preferred promoter is plac (with a laci^(q) on the vector toreduce background expression), which can be regulated by the addition ofisopropylthiogalactoside (IPTG). A second preferred promoter is pT7φ10,which is specific to T7 RNA polymerase and is not recognized by E. coliRNA polymerase. T7 RNA polymerase, which is resistant to rifamycin, isencoded on the defective lambda DE lysogen in the E. coli BL21chromosome. T7 polymerase in BL21(DE3) is super-repressed by thelaci^(q) gene in the plasmid and is induced and regulated by IPTG.

Typically, a culture of transformed bacteria is incubated with theinducer for a period of hours, during which the synthesis of the proteinof interest is monitored. In the present instance, extracts of thebacterial cells are prepared, and the chaperonin polypeptides aredetected, for example, by SDS-polyacrylamide gel electrophoresis. Afterthe E. Coli have been given sufficient time to produce enough protein,the protein is isolated and purified.

The expression of the chaperonin polypeptides in E. coli allows forsynthesis of large quantities of the proteins and also allows for theexpression and in some cases the assembly of different components in thesame cells. The methods for scale-up of recombinant protein productionare straightforward and widely known in the art, and many standardprotocols can be used to recover the wild-type and mutated chaperoninpolypeptides from a bacterial culture.

Purification of the chaperonin subunits can be using standard methods.In one non-limiting example, a purification procedure comprises, eitheralone or in combination: 1) chromatography on molecular sieve,ion-exchange, and/or hydrophobic matrices; 2) preparativeultracentrifugation; and 3) affinity chromatography.

In an embodiment where the chaperonin polypeptides are thermostableextremophiles, the cell extracts can be heated for easier purificationof the subunits. For example, the purification of the chaperonin betasubunit of Solfolobus shibatae expressed in E. coli involves heatingtotal cell extracts to 85° C. for 30 minutes, which precipitates most E.coli proteins, but the thermostable beta remains soluble. Therefore,heating and centrifuging cell extracts separates the beta subunit frommost E. coli proteins, which simplifies further purification using ionexchange chromatography (Kagawa, H. K. et al., 1995, The 60 kDa heatshock proteins in the hyperthermophilic archaeon Solfolobus shibatae. JMol Biol 253, 712-25).

In one embodiment, several different types of chaperonin polypeptidescomponents can beco-expressed in the same bacterial cells. Someassemblies of the polypeptides into chaperonins or higher orderstructures from purified wild-type or genetically modified subunits areextracted subsequent to limited in vivo assembly, using the methodsenumerated above. An example of a higher order structure is ananotemplate.

For forming the double-ringed structures of the chaperonins (and as seenlater, the nanotemplates) of the present invention, the purifiedsubunits are combined in vitro with Mg²⁺ and ATP, ADP, AMP-PNP, GTP orATPγS. In an alternate embodiment, purified chaperonins are assembled invitro with Mg²⁺ and ATP, ADP, AMP-PNP, GTP or ATPγS into thenanotemplates. In yet another embodiment, mixtures of purified subunitsand purified chaperonins are combined in vitro with Mg²⁺ and ATP, ADP,AMP-PNP, GTP or ATPγS to form the nanotemplates. The temperature or pHfor formation will depend on the type and thermostability of thechaperonin polypeptides or chaperonins. For example, for thethermostable chaperonin beta subunit of S. shibatae, the temperature canbe 75° C.-85° C., while it may be lower for other types of polypeptides(e.g., less than 40° C.). For a given chaperonin polypeptide, ornanotemplate, optimal conditions for assembly (i.e., concentration andproportion of Mg²⁺ to ATP, ADP, AMP-PNP, GTP or ATPγS) are easilydetermined by routine experimentation, such as by changing each variableindividually and monitoring formation of the appropriate products. Forformation of the chaperonins and/or nanotemplates, any of Mg²⁺, ATP,ADP, AMP-PNP, GTP or ATPγS can be present in an amount ranging from 1mM, up to 10 mM, 20 mM, 30 mM or higher. See, e.g., Yoai et al., 1998,Archives of Biochemistry and Biophysics 356, 55-62, where filaments areformed in 5 mM Hepes buffer with 25 mM MgCl₂ and 1 mM ATP (total volume300 μl). While it has been shown that the formation of chaperoninsand/or nanotemplates from α and β subunits of S. shibatae does notdepend on the presence of K⁺, formation of the higher order structuresfrom the subunits of other organisms may require the presence of K⁺.

In yet another embodiment, the chaperonins or nanotemplates are formedin the absence of introduction of any of Mg²⁺, ATP, ADP, AMP-PNP, GTP orATPγS. At sufficiently high concentrations of the chaperonins, e.g., atconcentrations of 2-5 mg/ml, or up to 30 mg/ml or more, some of thehigher order structures, such as the nanotemplates, can spontaneouslyassemble (Quaite-Randall et al., 1995, J. Biol. Chem. 270, 28818-28823).The concentration of the chaperonins or chaperonin polypeptides indifferent embodiments is 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2, mg/ml, 5mg/ml, 10 mg/ml, 30 mg/ml, 50 mg/ml or higher.

Alternatively, one or more extracts, for example crude bacterialextracts, containing the chaperonin polypeptides may be prepared, mixed,and assembly reactions allowed to proceed prior to purification.

In specific embodiments, some combination of both group I and group IIchaperonins and/or chaperonin polypeptide subunits can be mixed andallowed to assemble in vivo or in vitro.

In another embodiment, the product of the expression of the chaperoninpolypeptide and the resulting chaperonin is substantially free of other(non-chaperonin) proteins.

The methods and formulation conditions described herein for theformation of chaperonins can also be applied for the formation of thethe compositions and devices, e.g., the nanotemplates, nanostructures,nanodevices and nanoarrays ,of the invention due to the ability of thechaperonin and chaperonin polypeptides to self-assemble under suchconditions into the higher order structures.

Nanotemplates

The present invention provides methods for exploiting the subunits ofchaperonins to form nanotemplates. A nanotemplate comprises one or morechaperoning, wherein at least one chaperonin is a mutant chaperonincomprising at least one mutated polypeptide. As such, a nanotemplate cancomprise any number of chaperoning, in any proportion of mutant andwild-type chaperoning. In a non-limiting embodiment, the nanotemplate isa composite of only mutant chaperoning. The chaperonins comprising thenanotemplate can be group I chaperoning, group II chaperoning, or somecombination thereof. In different embodiments, the nanotemplatecomprises eukaryotic TCP-1, thermal factor 55, thermal factor 56 orGroEL chaperoning. In a preferred embodiment, the nanotemplate comprisesHSP60s and variants. The choice of chaperonins to comprise thenanotemplate can be made depending on factors such as operatingconditions. In a specific embodiment, if the nanotemplate is toexperience high operating temperatures, the one or more chaperonins canbe formed from extremophiles. The one or more chaperonins forming thenanotemplate can comprise 7, 8 or 9 subunits per ring, corresponding toseven-fold, eight-fold or nine-fold symmetric chaperoning, respectively.The chaperonins can comprise different types of subunits, for example,alpha subunits, beta subunits, gamma subunits or any combinationthereof.

In one embodiment, the nanotemplates of the present invention are formedfrom chaperonin subunits and/or chaperonins combined in vitro with Mg²⁺and ATP, ADP, AMP-PNP, GTP or ATPγS. In an embodiment, assembly mayrequire the presence of K⁺. In an alternate embodiment, thenanotemplates spontaneously self-assemble, and are formed fromchaperonin subunits and/or chaperonins combined in vitro in the absenceof introduction of any of K⁺, Mg²⁺, ATP, ADP, AMP-PNP, GTP or ATPγS. Atsufficiently high concentrations of the chaperoning, e.g., atconcentrations of 2-5 mg/ml, or up to 30 mg/ml or more, some of thehigher order structures, such as the nanotemplates, can spontaneouslyassemble (Quaite-Randall et al., 1995, J. Biol. Chem. 270, 28818-28823).The nanotemplate can be formed from chaperonins or chaperoninpolypeptides at a concentration of 0.1 mg/ml, 1 mg/ml, 2 mg/ml, 5 mg/ml,20 mg/ml or higher.

The length of filaments, one type of nanotemplate, can be manipulatedaccording to whether ATP, ADP, AMP-PNP, GTP or ATPγS is used in formingthe filament. Use of ATP, for example, can result in an extensivenetwork of filaments, while using ADP, AMP-PNP, GTP or ATPγS can resultin the formation of shorter. For example, with respect to formation ofnanotemplates comprising mutant and/or mutant and wild-type TF55 α and βsubunits of S. shibatae, chaperonins can be formed at concentrations ofapproximately 0.1 mg/ml, while at approximately 0.5 mg/ml, filamentsaare formed. Longer aligned filaments can be formed at concentrations ofapproximately 1.0 mg/ml. . See, also, for example, Yaoi et al., 1998,Archives of Biochemistry and Biophysics 356, 55-62, and Trent et al.,1997, Proc. Natl. Acad. Sci 94, 5383-5388 for examplary conditions thatcan be used to form structures like filaments of differing averagelengths or two-dimensional arrays. Thus, the length of filaments can becontrolled through manipulation and choice of formation conditions, withexact concentrations necessary for particular structures being routinelyattainable.

The nanotemplates can have different architectural symmetries, which canbe dictated through varying the formation conditions, or throughdirected binding or arrangement of the chaperonins relative to eachother. As a result, the nanotemplate can have one-, two-, three-, four-,five-, six-, seven-fold architectural symmetry. Chaperonins of theinvention can, for example, be used own to form nanofilaments (ananotemplate with one-dimensional architectural symmetry) in thepresence of Mg²⁺ and nucleotides. These nanofilaments can cluster toform bundles of filaments that are microns in length and with bundlediameters of up to microns in thickness.

FIG. 5 shows that in the electron microscope individual HSP60s in thedouble-rings appear as black “blobs” (A, end view) or alternating darkand light bands (B, side view). These double-rings self-assemble intochains or porous tubes (C) and the tubes associate into filaments (D).FIG. 6 shows the organization of HSP60 rings into 2-dimensional crystalson a metal grid coated with lipid (A) and filament bundles arranged on abed of rings (visible as spots in background) (B). In general, thechoice of proportion of ATP to Mg²⁺ affects the structure of theresulting chaperonin and nanotemplate, in terms of whether it forms afilament or an array. The nanotemplate can have long range two- orthree-dimensional ordering as in an array with trigonal or hexagonalclose packed architectural arrangement of the chaperonins throughself-assembly (FIG. 6), as described in greater detail below.

The various architectural symmetries can also be dictated throughdirected arrangement of the chaperonins onto a substrate either througha masking technique or by directed binding (Whaley et al., 2000, Nature405, 665-668, which describes peptides that bind to selectively tospecific faces gallium arsenide, silicon or indium phosphide). Anexemplary, non-limiting list of partial amino-acid sequences from clonesthat bind to different surfaces of GaAs and/or InP (Whaley et al., 2000,Nature 405, 665-668) includes: VTSPDSTTGAMA (SEQ ID NO:16) AASPTQSMSQAP(SEQ ID NO:17) AQNPSDNNTHTH (SEQ ID NO:18) ASSSRSHFGQTD (SEQ ID NO:19)WAHAPQLASSST (SEQ ID NO:20) ARYDLSIPSSES (SEQ ID NO:21) TPPRPIQYNHTS(SEQ ID NO:22) SSLQLPENSFPH (SEQ ID NO:23) GTLANQQIFLSS (SEQ ID NO:24)HGNPLPMTPFPG (SEQ ID NO:25) RLELAIPLQGSG (SEQ ID NO:26)

Whaley et al. also describes amino-acid sequences that bind silicon andnot silicon dioxide. An example of an amino-acid sequence that binds toZnS(102) (Lee et al., 2002, Science 296, 892-895) is: CNNPMHQNC (SEQ IDNo:27)

A list of partial amino-acid sequences from clones that bind to Ag (Naiket al., 2002, Nature Materials 1, 169-172) includes: AYSSGAPPMPPF (SEQID NO:28) NPSSLFYRLPSD (SEQ ID NO:29) SLATQPPRTPPV (SEQ ID NO:30)

A list of partial amino-acid sequences from clones that bind to Au(Brown et al., 2000, J. Mol. Biol. 299, 725-735; Brown, S, 1997, NatureBiotechnol. 15, 269-272) includes: MHGKTQATSGTIQS (SEQ ID NO:31)ALVPTAHRLDGNMH (SEQ ID NO:32)Other nanotemplates possessing shorter-range ordering include nanoringswith rectangular, pentagonal, hexagonal or heptagonal architecturalarrangements of chaperonins.

In a specific embodiment, the nanotemplate can comprise one or morewild-type and/or mutant chaperonins which serve as “spacers” in thenanotemplates. The spacer chaperonins can be confined to specificregions of the nanotemplate, and would not present specific bindingsites for any of polypeptides, nanoscale materials or linker molecules.The spacers can therefore serve a similar function as a mask insemiconductor fabrication.

The generation of several different mutations of a given subunit canresult in differences in dimension of the resulting cheronins thatcomprise the nanotemplate. For example, a variant produced through theremoval of a 28 amino acid loop at the apical end from of the β subunitof S. shibatae resulted in a chaperonin with an expanded internal porediameter of from 2.5 nm to °9 nm (see FIGS. 7B-D). This can be exploitedin forming a nanotemplate with different mixtures of chaperonin subunitvariants to present pores with different pore diameters for the bindingof nanosale objects such as nanoparticles and/or quantum dots.

The chaperonins and/or nanotemplates can differ according to the typesof subunits and also the combinations of types of subunits used information. For example, in vitro alpha and beta subunits of S. shibataeform homo-oligomeric rosettasomes, while mixtures of alpha, beta, andgamma form hetero-oligomeric. It has also been found that betahomo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes ofS. shibatae associate into filaments. FIG. 15 shows the protein sequencealignment of S. shibatae TF55 alpha subunit (SEQ ID NO: 39), betasubunit (SEQ ID NO: 1) and gamma subunit (SEQ ID NO: 38). In vivorosettasomes are hetero-oligomeric with an average subunit-ratio of1α:1β:0.1γ in cultures grown at 75° C., a ratio of 1α:3β:1γ in culturesgrown at 60° C., and a ratio of 2α:3β:0γ after 86° C. heat shock.Additionally, it has been observed that rosettasomes containing gammawere relatively less stable than those with alpha and/or beta subunits.A protein sequence alignment of the alpha, beta, gamma subunits of S.shibatae (see Figure), also provides useful information for positioningmutations on the chaperonin polypeptides. FIGS. 16A and 16B provide theDNA and amino-acid sequences of isolated S. shibatae TF55-γ.

The isolated chaperonin polypeptide subunits from a given organism canassemble into different types of nanotemplates and other higher orderstructures (Kagawa et al., 2002, Molecular Microbiology, in press). Theisolated S. shibatae TF55 alpha subunit (SEQ ID NO: 39) alone formsdiscrete homo-oligomeric rosettasomes with the characteristic nine-foldring member symmetry, and arrays of rosettasomes. The isolated S.shibatae TF55 beta subunit (SEQ ID NO: 1) forms filaments ofrosettasomes and bundles of filaments. The isolated S. shibatae TF55gamma subunit (SEQ ID NO: 38) does not assemble into rosettasomes, butforms amorphous aggregates and non-uniform round objects. Were seen inthe TEM (FIG. 6C). Varying the proportions of the different subunitsfrom a given organism can also result in the assembly of differenthigher order structure being formed(Kagawa et al., 2002, MolecularMicrobiology, in press). A 1:1:1 mixture of S. shibatae TF55 alpha,beta, and gamma subunits results in heterooligomeric rosettasomes andfilaments that were less bundled than the ones formed from isolated betasubunits. The 1:1 mixture of S. shibatae TF55 alpha and beta subunitsresults in filaments that are indistinguishable from filaments formed bythe 1:1:1 mixtures of alpha, beta and gamma.

In one embodiment, the higher order structures, such as thenanotemplates and nanostructures, comprise at least one isolated S.shibatae TF55 gamma subunit. This embodiment of the invention cancomprise mutated or wild-type chaperonin polypeptides. In a specificembodiment, the higher order structures, comprise at least one isolatedS. shibatae TF55 gamma subunit and wild-type chaperonin polypeptides.

In another embodiment, the nanotemplate forms part of a coating or ananofabric. Due to the capability of the chaperonins to self-assemble inan ordered arrangement on a fairly large length scale as compared totheir pore diameters, they can be applied in these areas that could takeadvantage of the capability. Additionally, the resulting coating ornanofabric can be made to include optical, electric, magnetic,catalytic, or enzymatic moieties as functional units. These are producedthrough the selected placement of different nanoscale materials theapical domain of the chaperoning e.g., near the pores of thenanotemplates, or on other binding sites of the chaperonin, or inbetween chaperoning. The inclusion of nanoscale material with thenanotemplates is discussed further in the section on nanostructures.

Changes in the subunit composition that can influence volume andreactivity of the central cavity of a chaperonin can also be exploitedfor various applications of the nanotemplates. While not wishing to belimited to a particular theory or mechanism, it is noted that the N- andC-termini of chaperonin subunits are believed to project into andocclude the central cavity. As such, because these termini can differbetween subunits of a given species (e.g., rosettasome of S. shibatae),changes in subunit composition of the chaperonin can be used to impacton the central. Changes in the volume and binding properties of thecentral cavity of the chaperonin can therefore be dictated based on thecomposition of the chaperonin, which can be exploited in the formationof nanostructures which present different types of binding sites fornanoscale materials. In certain embodiments the N- and C-termini aredeleted.

The assembly of chaperonin polypeptides, for example HSP60s, into suchstructures as rings, tubes, filaments, and sheets (2-D crystals) can beregulated chemically. The assembly can be manipulated by, for example,the proportion of ATP/Mg²⁺ and/or by manipulating the concentration ofthese regions. HSP60-rings, tubes, and filaments can, for example,function as nano-vessels if they are able to absorb, retain, protect andrelease gases or chemical reagents, including reagents of medical orpharmaceutical interest. On a nanoscale, the filamentous structures,preferably HSP60 structures, are hollow and chemicals that are diffusedor bound inside can be bound or released under programmed conditions attargeted locations.

The structures, e.g., rings, tubes, and filaments, can be induced toform ordered structures on surfaces. Under controlled conditions thechaperonins are observed to form 2-dimensional crystals on surfaces andthe filament bundles may be oriented on surfaces. In an alternateembodiment, the nanotemplate functions as a multi-nanowell assay plate,or a single-molecule probe for DNA detection and hybridization.

Layers of interwoven chaperonin filaments may form a nano-fabric. Suchfabrics may be induced to form on lipid layers and may ultimately beused to coat surfaces of materials. This may be of value in medicaltransplants in which the material could be coated with, e.g., an HSP60fabric from the host and thereby limit the immune response against thetransplant.

Fabrics or two-dimensional crystals of chaperonins comprising HSP60 canform nano-arrays of DNA or RNA by taking advantage of the intrinsicaffinity of HSP60s for nucleic acids. Such arrays would represent anunprecedented density of DNA probes and thereby greatly amplify thedensity of information per unit area. Other kinds of probes based onother molecules that associate with HSP60 can also be developed.

For characterization, electron microscopy and electron probing methods(EDAX) can be used for investigating the contents of nano vessels, thecontinuity of nano-wires, the product of template experiments, and thenature of nano-fabrics. Atomic force microscope (AFM) can be used inimaging and analyzing features of these nanotemplates. The DNAnano-arrays can be tested by hybridization methods.

Nanostructures

The present invention provides methods for forming nanostructures. Thechaperonins offer many advantages over other molecules for thecontrolled assembly of complex architectures, in their ability toself-assemble. A nanostructure can be formed from a selective placementprocess involving self-assembly, or directed binding, depending on thedesired resulting architectural arrangement. The steps in the formationof a nanostructure can include adding one or more nanounits comprising(i) at least one nanotemplate, (ii) at least one wild-type chaperonin,or (iii) a mixture of (i) and (ii) to a surface, and adding one or morenanounits comprising (i) at least one nanoparticle, (ii) at least onequantum dot, or (iii) a combination of (i) and (ii) to said surface. Anyunbound nanounits are removed in order to maintain the desiredarchitecture. Each of the addition steps are repeated as many times asnecessary to result in a nanostructure. Optimal conditions for assembly(i.e., concentration and proportion of Mg²⁺ to ATP, ADP, AMP-PNP, GTP orATPγS) are easily determined by routine experimentation, such as bychanging each variable individually and monitoring formation of theappropriate products. In alternate embodiment, the nanostructuresassemble in the absence of any of Mg²⁺, ATP, ADP, AMP-PNP, GTP or ATPγS.In yet other embodiments, assembly may require the presence of K⁺.

The resulting nanostructures utilize proteins to control the assembly ofstructures that may, in certain embodiments, incorporate organicmaterials or inorganic materials such as metallic, semiconducting ormagnetic nanoparticles (Bruchez et al., 1998, Science 281, 2013-16; Penget al., 2000, Nature 404(6773), 59-61; Whaley et al., 2000, Nature 405:665-68).

For the formation of a nanostructure, nanoscale materials can becombined with the chaperonin polypeptides and/or chaperonins undersuitable conditions (e.g., concentration and proportion of Mg²⁺, K⁺,ATP, ADP, AMP-PNP, GTP or ATPγS). The nanoscale material (i.e., thenanoparticle or quantum dot) can be attached to the chaperonin and/orthe polypeptide subunits at specific binding sites prior to assembly ofthe nanostructure. The nanoscale materials can be introduced before theformation of the nanotemplates, e.g., by being directly bound to asubunit, prior to assembly of the various subunits and/or chaperoninsinto the nanostructures. In an alternate embodiment, the nanoscalematerial is attached to specific binding sites after the nanotemplate isassembled. In such an embodiment, a nanotemplate is first formed, withthe selected sites for binding of the nanostructures present onpre-determined locations of the nanotemplates, and then thenanostructures are introduced.

In another embodiment, the nanoparticles are coated with a coating thatallows specific binding of the nanostructures to the pre-determinedlocations on the nanotemplates.

FIG. 10A shows a gold particle derivatized with surface-accessible,thiol-reactive maleimide groups (monomaleimido Nanogold, Nanoprobes,Inc.). The nanogol quantum dots were covalently bound to the mutant betasubunit of S. shibatae with a cysteine presented as a binding site.

In other embodiments, the nanoscale materials are coated with aninorganic and/or organic compounds, a polymer, a protein, a peptide,hormones, antibodies, nucleic acids, receptors, reactive chemicalgroups, binding agents and the like. For example, the nanoscalematerials can be coated with a polyethylene glycol compound containingchemically reactive amine groups.

In yet another embodiment, the nanoscale materials are coated withbiotin or streptavidin. In a specific embodiment, the nanoscalematerials are coated with bovine serum albumin (BSA) and biotin, and thestreptavidin is located at one or more binding sites of thenanotemplate. In another example, amino acids, or small peptides arecoated directly on the surface of the nanoscale materials, or arechemically linked to polymers or other type of macromolecules.

Examples of nanoscale materials include, but are not limited to,nanoparticles. such as gold, silver and other metal nanoparticle orcomposite nanoparticles of the metals; quantum dots (QD), includingCdSe—ZnS, CdS, ZnS, CdSe, InP, InGaAs, CuCl, and InAs quantum dots,silicon nanocrystals and nanopyramids, silver nanoparticles; or magneticquantum dots, e.g., nanomagnets, such as CoCu, FeCu, NiFe/Ag, and CoAgnanomagnets. The nanoscale materials can comprise one or more materials,or combinations of materials, such as transition metals, including gold,silver, zinc, cadmium, platinum, palladium, cobalt, mercury or nickel;alkali or alkaline earth metals, including sodium, potassium, calcium orcesuim; group III elements, including, aluminum, gallium or indium;group IV elements, including, silicon, germanium, tin or lead; group Velements, including, phosphorous, arsenic, antimony, or bismuth; orgroup VI elements, including, sulfur, selenium or tellurium. The listedmaterials can be in any given combination. Examples of III-V compoundsinclude GaAs or AlGaAs. The nanoscale material could also be afullerene, a carbon nanotube, or a dielectric, polymeric, orsemiconducting nanoparticle. In an alternate embodiment, flexibleprotein joints may be added to rigid carbon nanotubes to increase thediversity of possible forms while maintaining the functional featuresinherent in both kinds of nano-structures.

The size of the nanoscale material can be about 0.5 nm, 1 nm, about 10nm, about 50 nm, about 100 nm, about 200 nm, or about 500 nm, or more.The size of the nanoparticles can depend on the location of the bindingsite on the nanotemplate. If the binding site is at an apical domain, orwithin the internal cavity of the chaperonin, then the size of nanoscalematerial may correlate with the pore diameter of the chaperonin to whichit binds. FIG. 7C and 7E show that the size of the nanoscale materialthat bind at the apical domain of chaperonins formed from variants ofthe beta subunits of S. shibatae. FIG. 7C shows an illustration of the3-nm-pore 2D crystal (p312) indicating how 5 nm gold binds within theengineered pores. FIG. 7E shows an illustration of the 9-nm-pore 2Dcrystal (p312) indicating how 10 nm gold binds within the engineeredpores. The nanoscale materials may also be located in interstitialregions of the nanotemplate, i.e., between the chaperonins. Thenanoscale materials may be bound to more than one chaperonin, such aswhen the nanoscale material in present in an interstitial site. Inanother embodiment, the nanoscale material is located on top of a regionof the nanotemplate, and serve as a type of “mask.” In this embodiment,the nanoscale material can range up to 500 nm in size.

Morphologies of nanoparticles include, for example, nanopillars,nanocrystals, nanorods, nanotubes, nanowires, nanofilaments, nanofibersand composite metal/dielectric nanoshells.

In a specific embodiment, application of an electric field is used todisrupt the nanostructure or the.

In an alternate embodiment, differing amounts or proportions of ATP,ADP, AMP-PNP, GTP or ATPγS are used to disrupt the nanostructure ornanotemplates, or to cause the nanoscale material to become unbound fromthe nanostructure or nanotemplate.

In an embodiment, amino acid tails that do not inhibit their ability toassemble into rings and tubes are attached to the chaperoninpolypeptides, e.g.i, HSP60s, and that allow the binding of the nanoscalematerials inside the chaperonins structure, at an apical, equitorial orintermediate domain, or on other locations of the chaperonin.

Mutated chaperonin polypeptides, including HSP60s, can form nanometer ormicron scale tubes and filaments or arrays containing metals or doped orundoped semiconductors, and could function as nano-wires, field-effecttransistors, switches, diodes or logic devices. Given that metals can beattached to chaperonin polypeptides, their assembly into tubes wouldcreate a protein coated metal-cored conduit, i.e., a wire. By orientingand networking such wires nano-circuitry can potentially be created,which may be of value in the computer industry.

The nanostructures can also be incorporated into coatings with optical,electric, magnetic, catalytic, or enzymatic moieties as functionalunits.

Nanoarrays

A nanoarray is a nanoscale or microscale ordered arrangement ofnanotemplates and/or nanostructures. A nanoarray, therefore comprises anordered array of nanostructures. A nanoarray can have any type of longrange packing symmetry, including 2-, 3-, 4-, or 6-fold packingsymmetry. The nanoarray can be a one-dimensional structure, atwo-dimensional array, or a three-dimensional array. In a specificembodiment, where the nanoparticles are dielectrics, a three-dimensionalnanoarray can be a photonic bandgap crystal. Optimal conditions forassembly and crystallization of a nanoarray (i.e., concentration andproportion of Mg²⁺ to ATP, ADP, AMP-PNP, GTP or ATPγS) are easilydetermined by routine experimentation, such as by changing each variableindividually and monitoring formation of the appropriate products.

In both classes of beta mutants of S. shibatae, the single nativecysteine residue in beta is changed to a nonreactive alanine to preventpotential problems with folding and with assembly of mutant subunits.The cysteine is then placed at different solvent-exposed sites. Thethiols of these cysteines provide binding sites for soft metalsincluding gold and zinc. In one class of beta mutants, the exposedcysteine was placed near the tip of a 28 amino acid loop on the apicaldomain of beta, which in the assembled chaperonin protrudes into thecentral cavity. FIG. 7A-E shows the assembly of engineered HSP60s intonanoparticle array templates of the preferred embodiment. This mutantchaperonin has a ring of reactive thiols with a diameter ofapproximately 3 nm on both ends (FIG. 7A, left). In the other class ofbeta mutants, the protruding 28 amino acid loop is removed and placedthe exposed cysteine on the apical domain itself. The mutant chaperoninassembled from this subunit has a ring of reactive thiols with adiameter of approximately 9 nm and an open pore into its central cavity(FIG. 7A, right). FIG. 7A(top left) shows a model of a mutated HSP60beta subunit indicating apical loop cysteine placement by an arrow. Theside view is consistent with both classes of chaperonin variantsassembled from mutated beta subunits into two symmetrically stackednine-fold rings (FIG. 7A, center), while FIG. 7A (bottom left) shows atop view of beta chaperonin variant revealing 3 nm pore ringed by ninecysteines.

The TEM image of a negatively stained 2D crystal of the beta chaperoninvariant with cysteines substituted into the apical pores is shown inFIG. 7B. The two-sided plane group p312 was assigned to the latticethrough image analysis of micrographs of beta chaperonin 2D crystalsfrom S. shibatae (Koeck et al., Biochim. Biophys. Acta 1429, 40-44).FIG. 7A (top right) Result of genetic removal of the 28 residue apicalloop of beta and substitution of cysteine at the site fusing theα-carbon backbone. Residue deletion choices were made based on thestructural data from the model in FIG. 7A (left) as indicated by thearrows. FIG. 7B (bottom right) shows a top view of chaperonin variantwith 9 nm pore ringed by cysteines. FIG. 7B shows the 2D crystal of9-nm-pore variant detailing apparent increase in pore size by the changein electron density within the negatively stained rings. Both sampleswere imaged at the same condenser defocus setting. The ordering of thecrystal is illustrated by the FFT of the image. FIG. 7C shows anillustration of the 3-nm-pore 2D crystal (p312) indicating how 5 nm goldbinds within the engineered pores. FIG. 7E shows an illustration of the9-nm-pore 2D crystal (p312) indicating how 10 nm gold binds within theengineered pores.

The beta subunit S. shibatae proves to have sufficient structuralplasticity in its apical domain to accommodate both the amino acidsubstitutions and deletions can be made without loss of its ability toform chaperonins and 2D crystals. Under reducing conditions both classesof beta mutants formed chaperonins that assembled into disk-shaped,hexagonally packed 2D crystals up to 20 μm in diameter (FIG. 7B, 7D).The order within the crystalline lattices is illustrated by fast Fouriertransformation (FFT) of the TEM images (FIG. 7B, inset) which producedan optical diffractogram expressing the periodicity.

To determine whether the thiol-containing 2D crystals of chaperoninsacts as templates to bind and order nanoparticle QDs into arrays,commercially available gold nanoparticles (Ted Pella, Inc, Redding,Calif.) of different diameters can be used (FIG. 8). FIG. 8 shows goldquantum dot binding to engineered chaperonins and chaperonin templates.The uniform dispersion of these gold QDs in aqueous solution allows themto bind to hydrated chaperonin templates. To increase their likelihoodof binding specifically to the reactive thiol of the cysteines; however,the nanoparticles can be passivated with the ligandbis(p-sulfonatophenyl)phenylphosphine (BSPP) (Loweth, C. J., Caldwell,W. B., Peng, X., Alivisatos, A. P. & Schultz, P. G. (1999) DNA-basedassembly of gold nanocrystals. Angew. Chem. Int. Ed. 38, 1808-1812).BSPP displaces the citrate shell formed during synthesis of gold QDs(Novak, J. P., Nickerson, C., Franzen, S. & Feldheim, D. L. (2001)Purification of molecularly bridged metal nanoparticle arrays bycentrifugation and size exclusion chromatography. Anal. Chem. 73,5758-5761) and thereby reduces nonspecific binding of the QDs to theprotein template. The passivated gold QDs were reacted with thechaperonin templates attached to formvar-coated TEM grids (see Example6.6) and imaged in TEM mode at 60 kV. At low magnifications thechaperonin 2D crystals were visualized in the TEM using the electrondensity of the gold QDs themselves. FIG. 8A shows a low magnificationTEM image of 10 μm diameter unstained 2D crystal of 9 nm chaperoninvariant with 10 nm gold QDs bound. Contrast is from gold QDs bound tothe crystalline lattice of the underlying protein template. Drying cancause significant cracking and contributes to distortions and separationof regions of order within the array. FIG. 8B Higher-magnificationstained TEM image of side views of 5 nm gold QDs tethered at the apicalpores of the 3-nm-pore mutant chaperoning. At high magnification thechaperonin-gold interactions were visualized in the TEM bynegative-staining samples with uranyl acetate. FIG. 8B (inset) shows aslab-view cutaway diagram of postulated orientation of 5 nm and 10 nmgold QDs bound at the apical pores of the two chaperonin variants. FIG.8C shows a stained image of 5 nm gold QDs bound within the pores of the3-nm-pore crystalline template. Occupied rings show the QDs (dark areas)surrounded and held in place by the outer protein density of thechaperonin pores. Empty rings have a brighter, less electron denseappearance. FIG. 8D shows ordered region of 10 nm gold bound to a9-nm-pore template with similar area coverage as in FIG. 8C. The proteinholding the QDs in place is more difficult to see due to the larger sizeof the 10 nm QDs. Individual chaperonins in solution were observed tobind gold QDs on one or both ends. The QDs are presumably held in placeby multiple dative bonds formed between the gold surface and the thiolswithin the pores (FIG. 8B).

In control experiments, using chaperonin 2D crystals without exposedcysteines and with or without the amino acid loop deletions, the goldQDs appeared randomly distributed with no specific binding to thechaperonin crystals. On the surface of chaperonin 2D crystals withcysteines, however, the gold QDs bound specifically onto the pores (FIG.8C) forming regions of order on the protein (FIG. 8D) separated from oneanother by the cracked regions that resulted from drying, indicatingthat the engineered chaperonin crystals function as templates for goldQDs in solution. These chaperonin templates were size selective whenattached to substrates and appeared to bind QDs only on the exposedside. Templates made from beta mutants with cysteines added to theapical loop that formed 3 nm rings of reactive thiols ordered 5 nm (+/−3nm) gold QDs, but did not order 10 nm (+/−2 nm) or 15 nm (+/−1 nm) goldQDs, which bound randomly on the template surface. Variations in sizedistribution of gold QDs are a result of the manufacturer's method ofsynthesis. The chaperonin templates with the loop removed and cysteineson the apical domains that formed 9 nm rings of reactive thiols ordered10 nm (+/−2 nm) gold QDs, but 5 nm (+/−3 nm) and 15 nm (+/−1 nm) QDsbound randomly. This size selectivity is due to the accessibility andpositioning of cysteine residues within the pores of the templates.

The precision of the center-to-center spacing of gold QDs ordered by thechaperonin templates was 16 nm (+/−2 nm, n=200) for both 5 and 10 nmgold QD arrays, as determined by TEM. This is consistent with thecenter-to-center spacing of the chaperonin pores in the underlyingtemplates. The edge-to-edge spacing between QDs ranged from 6 to 10 nmfor arrays made with 5 nm (+/−3 nm) QDs bound to 3-nm-pore chaperonintemplates and from 4 to 6 nm for arrays made with 10 nm (+/−2 nm) QDsbound to 9-nm-pore chaperonin templates. This variation in spacing canbe attributed to both the variation in the size of the gold QDs and toimperfections in the lattice of the chaperonin templates resulting fromdrying, cracking and dislocations within the arrays. The observedvariation in QD spacing could be decreased with improved routes to QDsynthesis having narrower size distributions. With more monodisperseQDs, the precision of center-to-center spacing in the gold nanoarraysshould make it possible to tune the physical properties of the arrays bycontrolling the interparticle coupling using different sized QDs(Dujardin, et al., 2002, Adv. Mater. 14, 775-788).

The chaperonin nanotemplate arrays can also bind and order semiconductorQDs to form nanoarrays. Quantum dots of size 4.5 nm luminescentcore-shell (CdSe—ZnS QDs) were used (Dabbousi, B. 0. et al. (1997)(CdSe)ZnS core-shell quantum dots: Synthesis and characterization of asize series of highly luminescent nanocrystallites. J. Phys. Chem. B101, 9463-9475). These QDs were reacted with 3-nm-pore chaperonintemplates attached to glass or formvar substrates. Semiconductor QDshave low solubility in aqueous solutions. A QD suspension intrioctylphosphine/trioctylphosphine oxide (TOP/TOPO) diluted withbutanol was reacted with dried chaperonin templates. Under theseconditions the QDs bound to the cysteine-containing chaperonin templates(FIG. 9), but not appreciably to chaperonin 2D crystals without exposedcyteines (FIGS. 12 and 13). This is consistent with observations that Znin the outer ZnS shell of CdSe—ZnS QDs binds solvent-exposed thiols(Chan, W. C. & Nie, S. (1998) Quantum dot bioconjugates forultrasensitive nonisotopic detection. Science 281,2016-2018).

FIGS. 9A-D show the semiconductor QD nanoarray of a specific embodinemt.FIG. 9A shows differential interference contrast (DIC) light micrographof an 8 μm crystalline disc of 3-nm-pore template with 4.5 nmluminescent CdSe—ZnS QDs bound. The differential interference contrast(DIC) image of the QD-bound template (FIG. 9A) and the correspondingfluorescent image reveal that QDs bound to cysteine thiol retain theirluminescent properties (Bruchez, M., Jr., Moronne, M., Gin, P., Weiss,S. & Alivisatos, A. P. (1998) Semiconductor nanocrystals as fluorescentbiological labels. Science 281, 2013-2016). FIG. 9B shows both dry andrehydrated discs fluoresced indicating the QDs bound to the surface ofthe template. Selectivity for cysteine is confirmed using 2D crystals ofbeta variant without added cysteines which showed minimal QD binding(supporting information), while FIG. 9C shows low magnification TEM ofan unstained array of CdSe—ZnS QDs. Image contrast is due to the boundsemiconductor QDs. The mottled appearance of both the QD luminescenceand the electron density of low magnification TEM images indicate thatthe QDs are unevenly distributed on the chaperonin templates. FIG. 9Dshows higher-magnification image of same crystal revealing an orderedregion of QDs bound to the protein lattice. At higher magnification ofunstained samples, regions of ordered QDs are visible. These regions areseparated by unoccupied regions where QDs did not bind to the proteintemplate. This difference could be due to drying or to solvent effectsof the butanol, both of which may alter the structure of the chaperonintemplate and the accessibility of the thiols. Water-soluble(silica-capped) CdSe—ZnS (Gerion, D. et al., 2001, “Synthesis andproperties of biocompatible water-soluble silica-coated semiconductornanocrystals,” J. Phys. Chem. B 105, 8861-8871) QDs containing exposedthiol groups can bind more uniformly to hydrated chaperonin templates.The thiols on these QDs, however, can cause them to aggregate, which canresult in the formation of defective arrays, in which case, it ispreferable that the thiols be removed.

Nanoscale materials can be maneuvered into nanoarrays and nanostructuresby first tethering them to chaperonin subunits and then ordered as thesubunits assemble into chaperonins and 2D crystals (nanoarrays) or othernanostructures. As an example, commercially available 1.4 nm gold QDsderivatized with surface-accessible, thiol-reactive maleimide groups canbe used (monomaleimido Nanogold, Nanoprobes, Inc., Yaphank, N.Y.). FIGS.10A-D show an embodiment of a nanogold nanoarray. FIG. 10A shows acovalent attachment of 1.4 nm monomaleimido Nanogold to subunits ofloop-minus beta variant of the beta subunit of S. shibatae throughMichael addition of cysteine thiol to QD surface maleimide groups. FIG.10A(right) shows possible arrangement of nine 1.4 nm covalently attachedNanogold QDs viewed at one end of a ring assembled from the derivatizedsubunit. FIG. 10B shows low magnification TEM image of a 2D crystallinearray lightly stained with methylamine vanadate. The dark circularfeature (arrow) demarks the analyzed area corresponding to thedashed-line spectrum in FIG. 10D and is the result of polymerization ofmobile hydrocarbon which is attracted to the beam periphery. FIG. 10Cshows higher-magnification brightfield EF-TEM image of the arrayrevealing the ordered pattern of electron density that extends acrossthe crystalline template. FIG. 10D shows XEDS spectra of bare carbonfilm (solid line) and Nanogold array (dashed line) from the probeoutlined in FIG. 10B. Characteristic X-ray peaks from gold (Au M_(α)˜2keV and Au L_(α)˜9.7 keV) confirm the presence of Nanogold within thearray and the relative absence of Au on the support film.

These Nanogold QDs were covalently bound to the mutant beta subunit withcysteine inserted in place of the 28 amino acid loop in the apicaldomain (FIGS. 10A-D). Subunits, with Nanogold attached, assembled intochaperonins in the presence of ATP/Mg²⁺ (FIG. 10A); these chaperoninsform 2D crystals (FIGS. 10B and 10C). The binding of the Nanogold QDsand localization within the pores of the chaperonin crystals wasconfirmed by analytical TEM (FIGS. 10 and 11A-11C). FIGS. 11A-11C showan HAADF STEM imaging of Nanogold array. FIGS. 11A-11C show the diameterof the features contributing to the array periodicity is consistent withmultiple QDs localized within each ring. The diameter of electrondensity observed within the chaperonin rings forming the array (FIGS.11A-11C) is approximately 8 to 12 times that observed for a single 1.4nm Nanogold QD (FIGS. 11A-11C). FIGS. 11A-11C show the periodicity fromthe Nanogold QDs localized within the rings extends across the entirecrystal.

Ordered hexagonally spaced inclusions within the crystalline templatewere observed and determined to contain gold by imaging methylaminevanadate stained Nanogold samples in brightfield Energy Filtering(EFTEM) mode and by using X-ray Energy Dispersive Spectroscopy (XEDS)(FIG. 10B-D). Oxygen plasma-treated carbon support films were usedbecause they are more stable in an electron beam than formvar. Becausethe protein templates do not adhere to plasma-treated carbon as well asto formvar, samples were stained with methylamine vanadate to enableidentification of their location on the substrate. The XEDS spectrum ofthe Nanogold array reveals distinct peaks due to gold that are wellseparated from vanadium and copper peaks from the stain andcarbon/copper support respectively (FIG. 10D).

High Angle Annular Dark Field (HAADF) Scanning/Transmission ElectronMicroscopy (STEM) was used to image the gold localized and orderedwithin the Nanogold arrays (FIGS. 11A-C). Comparisons of bare Nanogoldto Nanogold ordered into an array revealed that multiple Nanogold QDswere localized within the pores of the crystallized chaperonins (FIGS.11A and 11B). The HAADF image of the Nanogold crystal also confirms thepresence of gold within the chaperonin pores because contrast in HAADFimaging mode is atomic number dependent, and nearly independent of focusor thickness. An HAADF comparison of the diameter of bare Nanogoldparticles on carbon to the diameter of the gold nanoparticles containedwithin the central pores of the chaperonins that template the Nanogoldinto arrays reveals that the central diameters are approximately eightto twelve times that of the diameter of a single Nanogold QD. Thisobservation is consistent with a model which suggests that each ring cancontain up to nine Nanogold QDs (one per subunit). A lower magnificationHAADF image of a similar area of an array reveals the ordering of thegold extends throughout the template (FIG. 11C). High resolution XEDSmapping attempts of the gold within the array were unsuccessful as thecrystals were destroyed with the electron dose needed for suchmeasurements. EELS (Electron Energy Loss Spectroscopy) mapping using theAu O shell was correspondingly unsuccessful because the V M shell edgelies in close proximity to the Au O shell and thus masks the goldsignal. FIG. 12 shows a control experiment showing DIC (left) andfluorescent (right) images of non-cys-mutated chaperonin crystals afterincubation with CdSe—ZnS QDs. The luminescence intensity of thefluorescent image is barely visible indicating minimal QD binding. FIG.13 shows an Energy Filtered TEM thickness map of a typical 2D proteincrystal. The intensity in this image is the ratio of the inelasticsignal to the elastic signal and is proportional to the ration of t/λwhere lambda is the mean free path for inelastic scattering and t is thelocal mass thickness. Regions of nominally uniform intensity indicateregions of nominally constant mass thickness. Increasing intensityindicates increased thickness. At the various regions and at the edgesof the crystal one can observe clear transitions indicating that thecrystal is composed of several layers.

Crystal thickness measurements (AFM and TEM) suggest that these crystalscan be multilayered (supporting information), and are observed ascrystals ranging from 1 to 5 layers (approximately 20 to 200 nm). Theassembly of QDs into arrays by first covalently attaching them tosubunits may create more defect-tolerant arrays because each chaperoninis composed of 18 subunits and therefore there are 18 chances for eachsite in the array to contain at least one QD. Likewise, the regions ofQD ordering within arrays assembled this way appear to span thedimensions of the crystalline template and with fewer defects thanpreviously observed. These types of arrays may find use in applicationsthat demand longer range ordering than the 5 and 10 nm gold andsemiconductor nanoparticle binding protocols allow.

The invention thus provides a hybrid bio/inorganic approach to nanophasematerials organization where the functionality of proteins can berationally engineered. Using structural information and recombinantbiotechnology techniques, genetically engineered chaperonins can be madeto function both as nanotemplates and as vehicles for controllednanoscale organization of preformed QDs into ordered naoarrays, e.g.,arrays of nanomagnets. These nanotemplates, nanostructures, andnanoarrays can be “wired” together into functional nanodevices, forexample by using genetics, as alternate binding sites may be engineeredat different locations on the chaperonin.

Nanodevices

The possibility to induce asymmetry within the arrays by engineeringalternate facets of the protein crystal is exploited in forming thenanodevices of the present invention. A nanodevice comprises at leastone nanotemplate, at least one nanostructure, at least one nanoarray orsome combination thereof. A nanodevice can, for example, be anelectronic, semiconductor, mechanical, nanoelectromechanical, magnetic,photonic, optical, optoelectronic or biomedical device formed from atleast one nanostructure, at least one nanoarray, and/or at least onenanotemplate.

In a specific embodiment, the nanostructures are organized into ananodevice that functions with the chaperonins still present. In analternate embodiment, the chaperonins are removed before the functioningof the nanodevice. The nanotemplate and nanostructure provide anorganizational basis for attached molecules, nanoparticles and quantumdots. The attached nanoscale materials can be equally spaced at, e.g.,15 nm intervals, or selectively place at pre-determined sites. Takingadvantage of the fact that enzymes (such as proteases) can be used tospecifically remove the chaperonin, the nanotemplates can serve to leavebehind pure material accurately placed on a surface at nano-scaleresolution.

The steps in the formation of a nanodevice are similar to those forforming a nanostructure, except that the building blocks arenanotemplates, nanostructures, and/or nanoarrays. The steps can includeadding one or more nanotemplates, nanostructures, nanoarrays, or somecombination thereof to a surface, and then removing any unboundnanotemplates, nanostructures, or nanoarrays. The steps are repeated anydesired number of times, with the choice of material introduction beingchanged at each step to build the desired nanodevice. Other maskingtechniques, e.g., semiconductor fabrication can also be combined withthe present invention in the construction of the nanodevice.

There is no direct parallel of the present invention in thesemiconductor manufacturing industry. The use of protein-based templatesthat self assemble into highly ordered structures allow of theengineering of semiconductor materials on a size regime much smallerthan that currently attainable. Further, given the diversity of thechaperonin system (e.g, its ability to bind other biomolecules such aslipid and DNA/RNA) the compositions and devices of the invention canalso be utiilized in a biomedical, e.g., biomedical device, context.

The invention further provides methods to selectively depositnanoparticles or quantum dots in an ordered array onto inorganicsubstrates. DNA manipulation and genetic engineering of the genes thatcode for chaperonins can be used to generate specificity in molecularrecognition at defined sites within the protein. For example, byintroducing cysteine residues into the protein, it can specifically bindcolloidal gold molecules through dative bonding between the sulfhydryl(SH) moeity of Cys and AuO. This allows for the organization of goldnanoparticles into ordered arrays onto substrates. After organizing thegold onto the surface, the protein can be removed using a reactive ioncold plasma, leaving the patterned gold in place on a clean surface(FIG. 14), thereby producing a nanodevice of the invention. The HSP60sbound with proteins or peptides are capable of releasing the boundproteins or peptides in the presence of adenosine triphosphate (ATP) oracidic conditions (Udono and Srivastava, 1993, J. Exp. Med.178,1391-1396). xxx With advances in microbial genetics, for exampleusing phage and cell surface display to identify inorganic bindingpeptide sequences (Whaley et al., Nature 405, 665-668), the usefulnessof this system extends beyond soft metals to other materials by, forexample, the addition of sequences back into the loop region that wasremoved.

Examples of additional, non-limiting applications of the nanodevicesinclude field emitters, sensors, optoelectronic and all-opticalswitches, lenses, probes, lasers, nanoelectromechanical systems (NEMS),circuitry and nanoelectronics, nanomachines (e.g., by attachingnanomotors), neural networks (nanoelectrodes for connections),nanocomputers, quantum computers, high-density magnetic memory orstorage media, photonic crystals, nanocrystal antennas, multi-nanowellassay plates, nanocatalysts (e.g., palladium), nanopores forsingle-molecule DNA sequencing, amplifiers for telecomrnmunications(approximately 7 nm PbSe and PbS quantum dots have a tunable gap near1500 nm). Applications include, for example, memory or storage devices(e.g., hard-disk drive read heads, magnetic RAM), magnetic fieldsensors, magnetic logic devices, logic gates, and switches.

Further applications can also include, for example, biochipapplications. Quantum dots in a biochip, for example, can each accountfor at least one or several data bits. The position of a single electronin a quantum dot can attain several states, so that a quantum dot canrepresent a byte of data. In an alternate embodiment, a quantum dot canbe used in more than one computational instruction at a time.

Other applications of quantum dots include nanomachines, neuralnetworks, and high-density memory or storage media.

In an alternate embodiment, the nanodevice, nanotemplate or nanoarrayfunctions as a single-molecule probe for DNA detection, hybridization,and sequencing.

Polymer microspheres with uniformly embedded polymers have applicationsas, for example, active fluorescent building blocks in flat paneldisplays and luminescent labels in biological detection. Thisapplication is achieved by forming a nanodevice comprising a nanoarrayof embedded polymer nanoparticles Still further applications relate tomolecular motors, e.g., molecular motors in a biomedical context.

6. EXAMPLES Example 6.1

Models

A homology model for S. shibatae HSP60beta was made using the web-basedservice Swiss Model (http://www.expasy.ch/swissmod/SWISS-MODEL.html).Seven PDB entries of solved structures of homologous proteins were usedas templates scoring between 48% and 64% sequence identity in pairwisealignment with native S. shibatae beta. The structure was relaxed invacuo with the GROMOS96 force field. Symmetric operations werer appliedto the subunit to form nine-fold symmetrical rings which were assembledinto 18-mer chaperoning. All models were constrained by dimensionsobserved for different chaperonin views as measured in the TEM.

Example 6.2

Cloning and Sequencing of the Gamma Gene of S. Shibatae

The gamma gene was amplified by the polymerase chain reaction (PCR)method from S. shibatae genomic DNA purified using Qiagen Genomic Tips(Qiagen). PCR primers (P1: 5′-ATGAACTTAGAGCCTTCCTAT-3 (SEQ ID NO:33) andP2: 5′-TTAACTCCATAAGAAACTTGT-3′) (SEQ ID NO:34) were based on previouslypublished partial gamma sequence information (Archibald et al., 1999,Current Biology 9, 1053-1056). The inverse-PCR method (Ochman et al.,1988, Genetics 120, 621-623) was used to obtain the complete gamma geneand its flanking regions. Briefly, AseIdigested genomic DNA wascircularized by self-ligation and a 1.2 kbp fragment was obtained by PCRamplification after 25 cycles (30 sec at 94° C., 1 min at 50° C., and 1min at 72° C.), using Vent polymerase (New England Biol.abs). The PCRfragment was ligated into pBluescript SK(+) (Stratagene) to obtain aplasmid which was transformed into E. coli (strain DH5α). The gamma genewas sequenced on both strands by the dideoxy-chain termination method(Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467), andanalyzed using the program DNASTAR (DNASTAR, Inc.). FIGS. 16A and 16Bshow the DNA sequence (SEQ ID NO: 37) and amino-acid sequence for S.shibatae gamma subunit (SEQ ID NO: 38).

Example 6.3

Expression of the Gamma Gene of S. Shibatae in E. Coli

The complete gamma gene PCR was amplified from S. shibatae genomic DNAusing a pair of primers (Primer 1: 5′GAAAGAACATATGGCCTATTTATTAAGAGAAGGAACACAG-3′ (SEQ ID NO:35) and Primer 2:5′-TAAAGTACTCGAGAAAACCTAAATAAAATAATCATATCTTAAC-3′ (SEQ ID NO:36)). Thisfragment was cloned into the Nde I and Xho I sites of the plasmid vectorpET22b (Novagen). Expression in E. coli strain BL21(DE3) “codon plus” inLB media containing 50 mg/ml carbenicillin (Sigma) was under isopropylβ-D thiogalactopyranoside (IPTG) regulation. The alpha and beta geneswere similarly expressed (Kagawa et al., 1995, J. Mol. Biol.253,712-725).

Example 6.4

Genetic Modifications

A standard PCR mutagenesis method as described in Current Protocols inMolecular Biology was followed to introduce cysteine residues and todelete the portions of DNA coding for the apical loop. All mutantsubunits were purified as described in the text and in correspondingreferences.

Example 6.5

Chaperonin Assembly and Crystallization

Chaperonins were assembled from purified subunits with the concomitantformation of two-dimensional crystals in solution, without the need ofan interacting interface. Concentrated stock solutions of ATP and MgCl₂were added to purified subunits (1.5 mg/ml, 25 μM in 25 mM HEPES, 3.5 mMTCEP) such that the final ATP concentration is 4 mM and the final MgCl₂concentration is 10 mM. The crystallization solution was incubated at 4°C. overnight after which crystals are observed as a white precipitate.

Example 6.6

Quantum Dot Nanoarray Formation

For gold QD binding, crystalline protein templates were applied toformvar coated 200 mesh copper TEM grids and gold QDs were bound byfloating the sample-side of the grid on 5 μl drops of passivated QDsols, wicking away with filter paper and washing by floating on HATbuffer (25 mM HEPES, 0.1% sodium azide, 3 mM tris[2-carboxyethyl]phosphine hydrochloride, pH 7.5) for 10 minutes. This process wasrepeated up to 10 times as more applications increases the siteoccupation on the template. The 10 nm gold QDs bound better with fewerapplications than the 5 nm QDs. After 10 applications the 3-nm templateswere considerably broken up. Samples were viewed in a LEO 912 AB TEM at60 kV. All quantitative image analysis was performed using AnalySIS 3.5(Soft Imaging System Corp., Lakewood, Colo.).

Semiconductor QDs were bound and imaged in a similar manner as gold QDswith the exception that templates are applied to TEM grids, were washedwith water, dried and re-swelled with butanol before QD binding. Forlight microscopy, the crystals were applied to a formvar coated glassslide, rinsed with water, dried, rinsed with butanol, and covered with acoverslip. A dilute slurry of CdSe—ZnS QDs in TOP/TOPO/butanol waspassed over the crystals by capillary action and thoroughly rinsed withbutanol, and imaged in brightfield, DIC and fluorescence modes on aLeica DMR/X microscope.

Nanogold arrays were fabricated in the following manner. Subunits of theloopless mutant with the cysteine insertion are reacted with an excessof Nanogold as per the instructions supplied by the manufacturer. TheNanogold-tagged subunits were separated from unreacted protein andexcess Nanogold using gel filtration chromatography (BioRad BioGelP-10), concentrated to 1.5 mg/ml and assembled into rings and 2Dcrystals as described above.

Samples were applied to carbon coated grids that were briefly treatedwith an oxygen plasma to enhance protein adhesion to carbon. Specimenswere analyzed at room temperature using a double-tilt Be stage in a FEITecnaiF20 AEM. The instrument was operated in the Transmission (TEM),Scanning Transmission (STEM), High Angle Annular Dark Field (HAADF) andEnergy Filtering (EFTEM) modes at 200 kV using a Schottky Field EmissonGun (FEG) electron source. All X-ray Energy Dispersive Spectroscopy(XEDS) measurements weree made using an EDAX ultra thin window Si(Li)detector having a FWHM of ˜150 eV at Nm K_(α) while energy filtering andelectron spectroscopy was accomplished using a Gatan GIF2000 imagingelectron energy loss spectrometer. Nominal probe sizes used during thestudy varied between 0.5-500 nm, depending upon the nature of themeasurements/observations.

6. Miscellaneous

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A nanotemplate comprising one or more chaperonins, wherein at leastone chaperonin of said one or more chaperonins is a mutant chaperonincomprising at least one mutated chaperonin polypeptide.
 2. Thenanotemplate of claim 1, wherein said nanotemplate has 2-, 3-, 4-, 5-,6- or 7-fold architectural symmetry.
 3. The nanotemplate of claim 1,wherein said one or more chaperonins comprise a group I chaperonin. 4.The nanotemplate of claim 1, wherein said one or more chaperoninscomprise a group II chaperonin.
 5. The nanotemplate of claim 1, whereinsaid one or more chaperonins comprise a combination of group I and groupII chaperonins.
 6. The nanotemplate of claim 1, wherein said one or morechaperonins is a prokaryotic or archaeal chaperonin polypeptide.
 7. Thenanotemplate of claim 6, wherein said chaperonin polypeptides areextremophiles.
 8. The nanotemplate of claim 1, wherein said one or morechaperonins comprise 7, 8 or 9 subunits per ring.
 9. The nanotemplate ofclaim 1, wherein said one or more chaperonins comprise a wild-type ormutant GroEL.
 10. The nanotemplate of claim 1, wherein said one or morechaperonins comprise a wild-type or mutant eukaryotic TCP-1.
 11. Thenanotemplate of claim 10, wherein said one or more chaperonins possess8-fold ring symmetry or 9-fold ring symmetry.
 12. The nanotemplate ofclaim 10, wherein said one or more chaperonins comprise alpha subunits,beta subunits, gamma subunits, delta subunits, or some combinationthereof.
 13. The nanotemplate of claim 1, wherein said one or morechaperonins comprise a wild-type or mutant thermal factor 55 chaperoninpolypeptide.
 14. The nanotemplate of claim 13, wherein said chaperoninspossess eight-fold ring symmetry or nine-fold ring symmetry.
 15. Thenanotemplate of claim 13, wherein said chaperonins comprises alphasubunits, beta subunits, gamma subunits or some combination thereof. 16.The nanotemplate of claim 1, wherein said mutant chaperonin polypeptidecomprise one or more point mutations.
 17. The nanotemplate of claim 1,wherein said mutant chaperonin polypeptide comprise one or moresequences deleted.
 18. The nanotemplate of claim 1, wherein said mutantchaperonin polypeptide comprise one or more sequence insertions.
 19. Thenanotemplate of claim 1, wherein said mutated polypeptide comprisesdeletions or additions to the N- or C-terminus.
 20. A nanostructurecomprising at least one nanotemplate of claim 1 and at least onenanoparticle and/or at least one quantum dot.
 21. The nanostructure ofclaim 20, wherein said nanoparticle is a nanopillar, a nanocrystal, ananorod, a nanotube, a nanowire, a nanofilament, a nanofiber, ananocrystal, or a composite metal/dielectric nanoshell.
 22. Thenanostructure of claim 20, wherein said nanoparticle or quantum dotcomprises a metal.
 23. The nanostructure of claim 20, wherein saidnanoparticle or quantum dot comprises a semiconductor.
 24. Thenanostructure of claim 20, wherein said nanoparticle or quantum dotcomprises gold, silver, carbon, zinc, cadmium, silicon, gallium, lead,indium, platinum, palladioum, cobalt, mercury, arsenic or nickel. 25.The nanostructure of claim 20, wherein said nanoparticle is a polymericnanoparticle.
 26. The nanostructure of claim 20, wherein saidnanoparticle is a dielectric nanoparticle.
 27. The nanostructure ofclaim 20, wherein said quantum dot is a magnetic quantum dot.
 28. Thenanostructure of claim 27, wherein said magnetic quantum dot comprisescobalt, iron, copper, nickel, or silver.
 29. The nanostructure of claim28, wherein said magnetic quantum dot comprises a composite ofcobalt/copper, iron/copper, nickel/iron/silver, or cobalt/silver.
 30. Ananoarray comprising an ordered array of the nanostructures of claim 20.31. The nanoarray of claim 30, wherein said nanoarray has 2-, 3-, 4-, or6-fold packing symmetry.
 32. The nanoarray of claim 30, wherein saidordered array is a one-dimensional, two-dimensional array, or athree-dimensional array.
 33. The nanoarray of claim 30, wherein saidnanostructure is a nanopillar, a nanorod, a nanotube, a nanowire or ananofilament.
 34. The nanoarray of claim 30, wherein said nanoarray is aphotonic crystal.
 35. A nanodevice comprising at least one nanotemplate,at least one nanostructure, at least one nanoarray or some combinationthereof.
 36. The nanodevice of claim 35, wherein said nanodevice is afield-effect transistor, an integrated circuit, a field emitter, aprobe, a nanocomputer, a quantum computer or a sensor.
 37. Thenanodevice of claim 35, wherein said nanodevice is ananoelectromechanical system.
 38. The nanodevice of claim 35, whereinsaid nanodevice is an optoelectronic switch, an optical switch, a lens,a laser, photonic crystals, or an amplifier.
 39. The nanodevice of claim35, wherein said nanodevice is magnetic memory device, a magneticstorage device, a hard-disk drive read heads, a magnetic RAM, a magneticfield sensor, a magnetic logic gates, or a magnetic switch.
 40. Thenanodevice of claim 35, wherein said nanodevice is an electronic,semiconductor, mechanical, electromechanical, magnetic, photonic,optical, or optoelectronic device.
 41. The nanoarray of claim 30,wherein said nanoarray is a multi-nanowell assay plate, or asingle-molecule probe for DNA detection and hybridization.
 42. A coatingcomprising comprising at least one nanostructure of claim 20, at leastone nanoarray of claim 29, or some combination thereof.
 43. The coatingof claim 42, wherein said coating comprises optical, electrical,magnetic, catalytic, or enzymatic moieties.
 44. A method of forming ananostructure comprising: (a) adding one or more nanounits comprising(i) at least one nanotemplate, (ii) at least one wild-type chaperonin,or (iii) a mixture of (i) and (ii) to a surface; (b) adding one or morenanounits comprising (i) at least one nanoparticle, (ii) at least onequantum dot, or (iii) a combination of (i) and (ii) to said surface; (c)removing any unbound nanounits; and (d) repeating steps (a), (b) and (c)to form a nanostructure.
 45. The method of claim 44, wherein saidnanotemplate specifically binds said a least one nanoparticle or quantumdot.
 46. The method of claim 44, wherein said nanotemplate or wildtypechaperonin has at least one nanoparticle or quantum dot pre-attached.47. The method of claim 44, wherein said nanoparticle is of a sizebetween 1 and 500 nm.
 48. The method of claim 44, wherein said quantumdot is of a size between 1 and 500 nm.
 49. The method of claim 44,wherein said nanoparticle or quantum dot comprises a metal.
 50. Themethod of claim 44, wherein said nanoparticle or quantum dot comprises asemiconductor.
 51. The method of claim 44, wherein said nanoparticle orquantum dot comprises gold, silver, carbon, zinc, cadmium, silicon,gallium, lead, indium, platinum, palladioum, cobalt, mercury, arsenic ornickel.
 52. The method of claim 44, wherein said nanoparticle is apolymeric nanoparticle.
 53. The method of claim 44, wherein saidnanoparticle is a dielectric nanoparticle.
 54. The method of claim 44,wherein said quantum dot is a magnetic quantum dot.
 55. The method ofclaim 54, wherein said magnetic quantum dot comprises cobalt, iron,copper, nickel, or silver.
 56. The method of claim 55, wherein saidmagnetic quantum dot comprises a composite of cobalt/copper,iron/copper, nickel/iron/silver, or cobalt/silver.
 57. The method ofclaim 44, wherein said nanotemplate further comprises one or more pores.58. The method of claim 57, and wherein said at least one quantum dot ispresent inside said one or more pores.
 59. The method of claim 57, andwherein said at least one nanoparticle is present inside said one ormore pores.
 60. The method of claim 44, wherein said nanostructure isformed through self-assembly.
 61. The method of claim 44, wherein saidnanostructure comprises two or more nanotemplates or wild-typechaperoning, and wherein said nanostructure is formed through patterningsaid nanotemplates or wild-type chaperonins onto a surface.
 62. Themethod of claim 61, further comprising the step of removing saidnanotemplate, such that said at least one quantum dot and/or at leastone nanoparticle form a nanodevice.
 63. The method of claim 44, whereinsaid nanostructure forms a nanoarray.
 64. The method of claim 44,wherein said nanostructure forms a nanodevice.
 65. The method of claim44, wherein said nanostructure is formed in the presence of any of K⁺,Mg²⁺, ATP, ADP, AMP-PNP, GTP or ATPγS.
 66. The method of claim 44,wherein said nanostructure is formed the absence of any of K⁺, Mg²⁺,ATP, ADP, AMP-PNP, GTP or ATPγS.
 67. The method of claim 44, whereinsaid at least one nanotemplate, or at least one wild-type chaperonin areat a concentration of about 0.1 mg/ml, about 1 mg/ml, about 2 mg/ml,about 5 mg/ml, or about 20 mg/ml.
 68. An isolated polypeptide comprisingSEQ ID NO:1, wherein residue 298 is changed from cysteine to alanine.69. An isolated polypeptide comprising SEQ ID NO:1, wherein residue 270is changed from glutamine to cysteine.
 70. An isolated polypeptidecomprising SEQ ID NO:1, wherein residues 254 to 281 are deleted.
 71. Anisolated polypeptide comprising SEQ ID NO:1, wherein a peptide sequenceis inserted that possess binding specificity.
 72. An isolatedpolypeptide comprising SEQ ID NO:1, wherein a deletion or addition ismade to the N-terminus.
 73. An isolated polypeptide comprising SEQ IDNO:1, wherein a deletion or addition is made to the C-terminus.
 74. Amethod for forming a mutated chaperonin polypeptide comprising, (a)modifying at least one protein residue of a chaperonin polypeptide bypositioning a mutation to form one or more mutated chaperoninpolypeptides; (b) assembling said one or more mutated chaperoninpolypeptides to form a mutated chaperoning.
 75. The method of claim 74,wherein said assembly occurs in the presence of any of K⁺, Mg²⁺, ATP,ADP, AMP-PNP, GTP or ATPγS.
 76. The method of claim 75, wherein said K⁺,Mg²⁺, ATP, ADP, AMP-PNP, GTP or ATPγS are present in an amount of 1 mM,up to 10 mM, 20 mM, 30 mM or higher.
 77. The method of claim 74, whereinsaid mutated chaperonins spontaneously assemble to form said one or moremutated chaperonins.
 78. The method of claim 74, wherein said mutatedchaperonin polypeptides are assemble in vivo.
 79. The method of claim74, wherein said mutated chaperonin polypeptides are assemble in vitro.80. The method of claim 74, wherein said mutated chaperonin polypeptidesare at a concentration of 0.1 mg/ml, 1 mg/ml, 2, mg/ml, 5 mg/ml, 10mg/ml, 30 mg/ml, 50 mg/ml or higher.